STUDIES  ON  THERMOPHILIC  BACTERIA 


By 

LETHE  ELEANORA  MORRISON 

A.B.  University  of  Illinois,  1919 
M.S.  University  of  Illinois,  1921 


THESIS 

SUBMITTED  IN  PARTIAL  FULFILLMENT  OF  THE  REQUIREMENTS 
FOR  THE  DEGREE  OF  DOCTOR  OF  PHILOSOPHY  IN 
BACTERIOLOGY  IN  THE  GRADUATE  SCHOOL 
OF  THE  UNIVERSITY  OF  ILLINOIS,  1922 


URBANA,  ILLINOIS 


d»r  t z 


I 322 
M 8 3 

UNIVERSITY  OF  ILLINOIS 
THE  GRADUATE  SCHOOL 

August  15,  i92  2 


1 HEREBY  RECOMMEND  THAI'  THE  THESIS  PREPARED  UNDER  MY 

supervision  by Lethe  Eleanors  Morrison 

ENTITLED Studies  on  Thermophilic  Bacteria  


BE  ACCEPTED  AS  FULFILLING  THIS  PART  OF  THE  REQUIREMENTS  FOR 
THE  degree  of  Doctor  of  Philosophy 


Recommendation  concurred  in* 


Committee 


on 


Final  Examination* 


500412 


Digitized  by  the  Internet  Archive 
in  2016 


https://archive.org/details/studiesonthermopOOmorr 


Acknowledgement 


It  is  with  pleasure  that  the  author  takes 
this  opportunity  to  express  her  appr eolation  for  much 
kindly  advice  and  assistance  received  during  the  super- 
vision of  this  work  from  Professor  P#  W#  fanner#  She 
also  wishes  to  thank  him  for  encouragement  received  in 
all  graduate  study  and  teaching  done  under  his  supervision# 
Thanks  are  also  extended  to  those  others,  who 
by  various  means,  aided  in  the  completion  of  this  in- 
vestigation# 


4 . 


. 


. 


LIST  OF  TABLES  * 


Table  I 


Table  II 12 


Table  III 


20 


Table  IV 27 


■Table  V 29 


Table  VI 


29 


* m. 


Tables  follow  the  page  numbers  given  above* 


1 


I,  Introduction 

A new  interest  in  thermophilic  bacteria  is  rapidly- 
developing  since  the  investigations  of  Barlow  (1912),  Weinzirl 
(1919),  Cheyney  (1919),  Bonk  (1920),  and  Bigelow  and  Estey  (1920), 
have  suggested  their  significance  in  the  spoilage  of  canned  foods. 
The  wide  distribution  of  these  organisms  in  nature  is  demonstrated 
beyond  doubt  by  a review  of  the  literature  which  has  been  publish- 
ed describing  strains  isolated  from  soil,  water,  foods,  manure, 
ensilage,  hay,  cotton,  etc.  The  infection  of  foods  with  ther- 
mophiles  results,  probably,  from  their  contact  with  soil  and 
water.  The  object  of  this  investigation  was  to  study  the  charac- 
teristics, particularly  the  temperature  relations  including  ther- 
mal death  point  determinations,  of  some  thermophilic  bacteria 
isolated  from  different  samples  of  water  and  soil. 


i 


2 


II,  Historical 

In  order  to  have  the  present  dissertation  more  complete 
in  regard  to  the  historical  review  and  bibliography,  a table 
summarizing  the  literature  on  thermophilic  bacteria  used  in  the 
Master’s  Thesis  by  this  author  is  also  included,  (Table  I.) 


Since  the  experimental  work  of  this  investigation  deals 
principally  with  the  temperature  relations  and  thermal  death 
points  of  thermophilic  bacteria  isolated  from  various  sources, 
it  was  deemed  advisable  to  include  here  a brief  review  of  that 
literature  on  thermophilic  organisms  regarding  these  points  only. 


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Investigator 

Organism  Described 

Source 

•Temperature 

Remarks 

Mi  quel  (1879-99) 

Bacillus  therisophilue 

eine  River  water;  sewage 
xcreta;  dust;  nir 

20  -72°  C. 
15°  - 70°  G. 
jptimum 

Attributes  the  property  of  t ic  organism  to  grow  at  such  high  temperature  to 
particular  character  of  protoplasm.  Isol  -*d  first  in  1879  ; characteristics 
described  in  1388 

van  Tiornem  (1381) 

(1)  Streptococcus 

(2)  Bacillus 

;ter  in  which  beano  had 
ear.  cooked 

Jp  to  74°  C. 
jp  to  77°  C- 

A certain  umouht  of  acidity  produced  by  these  organisms  a.en  renderod  the  media 
uninhabitable  by  them 

Certoa  or.d  Garrigou 
(1986) 

(1)  Small  rods  f 

(2)  Filaments 

ot  epring  at  Luchon 

,5°  . 54°  c. 

"Further  experiments  are  necessary  to  determine  the  chemical  and  biological  a*ti  of 
these  rods,  and  this  knowledge  will  throw  a light  on  the  therapeutics  of  mineral 
waters" 

Globl g (1?93) 

Many  bacilli  (30  kinds) 

arden  soil 

j0°  - 70°  C. 

Thought  the  fact  that  ros  of  them  were  isolated  from  the  superfi:ial  layers  of  tho 
noil  explains  that  they  get  heat  for  high  temperature  at  which  they  grow  from  the 
rays  of  the  sun.  source  not  intestinal  t of  man  or  animals,  tap  or  river  -v.iber 

Eurrlli  (1339) 

Two  bacilli 

ilage ; manure 

60°  - 70°  C. 

Toe  initial  hig.  temperature  which  these  bacteria  induce  is  probably  most  6orviceuble 
by  causing  the  closer  packing  of  the  sil»ro  and  the  exclusion  of  the  air,  rather 
than  by  killing  the  germs  of  other  ferments 

Schloesing  (1989) 

table  manure 

Jp  to  79 .5°  C- 

At  temperature  of  60°  - 66°  C . these  organ!  -.3  produce  17  times  as  much  carbonic 
acid  as  that  in  sterilized  raanure. 

Cohn  (1893) 

Attributed  to  thermogenic  bacteria  a role  i.,  so-called  spontaneous  heating  of  r.alt, 
tobacco  leaves,  cotton,  hay,  and  manure-  Made  no  attempt  to  isolate  any  of  these 
cultures  by  the  plate  method. 

FI  Or  *9  (1394) 

Many  bacteria 

Sterile  milk 

4°  - 44°  C«  or 
27°  - 34°  C. 

All  strongly  peptonizing  in  character;  some  ere  toxic;  all  formed  spores  which 
would  withstand  heating  in  water  or  steam  for  two  hourr.. 

Griffiths  (1994) 

Bacillus  valericus 
Bacillus  therrdcus 

Silage 

50°  - 66°  C- 
56°  C*  optimum 

68°  - 680  c. 
55°  C*  opt i um 

Functioned  in  the  production  of  the  so-called  sweet  silage. 

Leichraan  (1894) 

A bacillus 

Slimy  milk 

45°  - 50’  C. 

Produced  acid 

UacFadyen  and  : 
(1894) 

Many  bacilli 

Earth,  river  and  sea  water 
river  mud,  air  dust,  straw 
and  feces  of  men,  mice  and. 
chickens 

60°  - 6SP 

"Their  most  marked  property  appears  to  be  the  decomposition  o-  proteid  codier.  • ich 
they  are  able  to  effect."  Most  of  them  possess  active  fermentation  properties. 

Gorihi  (1395) 

Milk 

37°  - 

Ambres  (1910)  suggested  that  this  organism  was  ther, ^tolerant  since  it  grew  at 
37<*  G • also  - 

Karlinskl  (199  5) 

(1)  Bacillus  IUidzensis 
capsulatue 

(2)  Bacterium  Ludwigi 

Hot  springs  of  Illidze  in 
Bosnia 

50°  - 58°  c. 
55°  - 57°  C. 

Has  no  explanation  to  offer  a3  to  significance  of  the  presence  of  these  two 
organisms  in  hot  springs.  Su^rests  that  the  examination  of  other  hot  springs  for 
similar  bacteria  would  greatly  increase  the  knowledge  of  biology  of  water  bacteria 

Rabinowitsch  (1895) 

Eirkt  specier.  of  thermophilic 
bacteria 

Many  sources;  widely 
distributed  in  nature 

3 4°  -75°  C. 

Concluded  that  peculiar  ability  of  so-called  thermophilic  bacteria  to  grow  at 
temperatures  so  much  higher  than  the  optimum  temperature  for  common  bacteria  is 
a property  of  adaptation  to  environment. 

Weter  (189  5) 

Bacillus  I 

Bacillu  II 
Bacillus  III 

"Sterile"  milk 

22°  - 50°  C. 
220  _ 50°  C. 
3 0°  - 650  c. 

Found  thermophilic  bacteria  in  eight  out  of  eleven  samples  of  so-called  "sterile" 
milk . T«o  of  the  three  bacilli  formed  sporec;  -.one  liquefied  gelatin;  all 

1 - 

T.-o  or  three  k i -.ds  of 
thermophilic  ncteria 

50«  . 60o  c. 

Mo  attempts  made  to  characterize  or  iden'ify  the  • orme  fo-  - 

Kedzoir  (1396) 

Cladothrix  form 

River  spree 

35°  - 65°  C. 

55°  C.  optimum 

Facultative  anaerobe  grows  better  without  oxygen.  Spores  very  resistant  to  heat 
p_nd  to  disinfectants  such  as  5 per  cent  phenol 

Teich  (1895) 

Bacillus  form 

Hot  springe  of  Illidze 

54°  - 58°  G. 

Large  oval  spores  formed  in  one  end  of  tie  rod*  make  then  pear  club-shaped 

Davis  (1897) 

Probably  a bacillus 

Hot  springs  of  Yellow- 
stem*  Park 

Up  to  85°  c. 

Functions  in  the  formation  of -miner  1 deposits  in  hot  springs. 

Harshbarger  (189  7) 

\Thite  filamentous  bacteria 

Hotsprihge  of  Yellow- 
stone Park 

35.4°  C. 

Eecomes  of  a sulphur  yellow  color  at  175°  F.  Yellow  '-.rowth  due  to  species  of 
Beggiatoa,  a plant  which  is  classed  with  the  Bacteriaceae , and  which,  during  life, 
deposits  sulphur  .granules- 

Koning  (1897) 

B.  tabaci  III 
B-  tabaci  IV 
B-  tabaci  V 

Tobacco 

Ambro*  (1910)  speaks  of  these  organisms  ns  real  thermophilic  bacVeria 

Uiyoshl  (1397) 

Many  bacillus  forms;  n zooglian 
mass  of  bacteria;  iron  bacteriui 

Hot  springs  in  Japan 

41-69.8°  C. 

Hot  springs  of  Japan  furnish  a good  medium  for  thermophilic  bacteria 

Wittlin  (1397) 

Hot  springs  in  Switzer- 
land 

Ambroz  (1910)  explains  the  negative  results  of  littlir.  on  the  grounds  of  inadequate 
methods  of  research. 

Laxa  (1398) 

Clostridium  gelatinosum 

F'Jllmass*  in  sugar 
manufacture 

:5°  - 58°  C. 

Tnis  organism  is  a facultative  anaerobe  whose  spores  are  not  killed  by  exposure  to 
steam  at  10CP  C.  for  15  minutes. 

Oprescu  (1898) 

Bacillus  thermophilus  lique- 
facien6  aerophilus 
Bacillus  thermbphilus  aerobius 
Bacillus  thermophilus  aquatilis 
Bacillus  thermophilus  reducens 
Eacillus  thermophilus  liquefac- 
iens  tyro genus 

Soil,  Berlin  zoological 
r ardens 
Canal  water 
Spring  water,  ice 
Blood  serum  test  gli-rs 
Roquefort  cheese 

21°  - 70°  C. 

36°  - 60°  C- 
36°  - 60°  C. 
36°  - 620  C. 
Room  tempera- 
ture to  60°  C. 

Oprescu  gave  rather  a detailed  description  of  the  five  forms  tudied  by  him 

Poupo  (1898) 

An  organism  simijiar  to  Clostri- 
dium gel  at  in  os  urn  Laxa  (1898) 

Syrup 

15°  C. 

Schillinger  (1898) 

Four  types 

Soil 

66°  C.  max. 

After  experiments  on  bacteria  from  soil  carried  on  t different  temperature  , he 
came  to  the  conclusion  that  thermophilic  bacteria  ore  not  properly  so-called;  the 
term  thermotolerant  should  be  applied  to  those  organisms  which  can  adapt  themselves 
to  high  temperatures. 

Teiklinsky  (1898  and 
1399) 

Theraoactinomyces  vulgaris 
Thermomyce3  lanuginocus 

Soil 

48°  - 68°  C. 

Also  isolated  6 varieties  of  bacteria  from  the  ot  sprines  of  Island  of  Ischia 
hich  she  called  strict  thermophiles;  0 timura  temperatiuee  60°  C- 

Michael  la  .(1839) 

B.  thermophilus  aquatilic 
liquefaciens 

B.  thermophilus  aquatilis 
liquefacie  s aerobius 
B.  thermophilus  aquatilis 
chromogenes 

VB.  Thermophilus  aquatilis 
anguinosus 

Spring  water,  Berlin 

50°  - 60°  C. 
optimum 

"ichaelis  said  of  thee,  organi  9 that  they  '-ere  not  only  thensotolerant,  1 ut  also 
thermophilic 

Vernhout  (1899) 

.cillus  tabaci  fermentationis 

Fermenting  tobacco  at 
4 i°  - 50°  0. 

50°  C«  optimum 

Mot  a true  ther  :iophile  cince  its  optimum  temperature  was  IS  C. 

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Invest! gator 

Organisms  Described 

Sources 

1 '^re 

Remarks 

?K.T,es  ( 10CO) 

Hino  err  nl  »i  not  named 

7uter,  feces,  coil,  pus, 
dlk,  etc. 

309  - 70°  0. 
c ptimum 

Suites  eu f roots  that  a distinction  be  made  between  "thermophilic"  and  "thormotolerant" 
bacteria. 

I 

Bacillus  thormophilus 

Gripooni 

onure 

30°  - "0"  C- 
••6°  C-  optiaUB 

- - - 1 • • 
The  organism  was  proteolytic  in  character. 

Russell  Hastings 

(1902) 

A micrococcus 

Pasteurized  milk 

200  . 25-  C. 
optimum. 

76°  C*  maximum 

' ut  a true  thermophile  : ut  probably  a tnersotolor&rt  organism. 

Schardinger  (1303) 

Group  I 6 aerobic  and  1 
anaerobic  orgar.ioms. 

Group  II  1 aerobic  and  5 ana- 
erobic organisms. 

~ooda, etc . 
i*oods,  etc. 

Rc 001-55®  C- 
37®  - 66° 

Grouped  with  h-  -rn  otato  bacilli,  produce  : Hj  fr,  r.  nitratee.  Found  that  these 
lacterir  "doxtri  ire"  starch. 

Setchell  (1903) 

Filamentous  schizomcete 

o-  Springs  of  Yellowstone 

Park 

70°  - 89°  C. 

Such  bacteria  grow  at  a higher  temperature  in  silicious  than  in  calcareous  waters. 

Tsiklir.sky  (1903) 

:.cilli.  TWO  streptc- 
thrix  forms 

Human  alimentary  tract 

57°  C- 

relieved  Ve  . ppearar.ee  of  tb.or. 

their  wide  distribution  in  nature  and  their  f£eat  resistance;  t ermophili-s  are 
probably  merely  variations  of  common  non-thermophilic  organisms* 

C.tterlna  (1904) 

Bacterium  thermophilus  radiat 

Y/ater 

60®  - 70°  c. 

optimum 

Also  isolated  an  organism  apparently  identical  with  B.  thermophilus  IS  (Rab'nowitsch, 
189  5)  A 

CilUrt  (1904) 

Actinomyces  therraop:  ilue 

Soil 

50®  - 55°  C- 
optimum 

Strict  aerobe;  liquefied  gelatin  slowly;  coagulated  milk. 

Kehler  (1904) 

Tvr  forms  , 

6 5®  C.  c;  *.i  aa 

. etti  ( 1905) 

A bacterium 

Hot  spring 

CO®  - 76°  C. 
o ptinum 

An  anaerobic  ram  positive  organism  v i h produced  large  central  spores. 

i rulni  (1905) 

Thirteen  bacilli,  five 
streptothrix  forms 

Adult  and  infant  stools. 

F ur  bacilli  an<  one  streptothrix  aboolute  thermophiles ; all  strict  aerobes;  all 
gram  positive;  all  but  one  6pore  formers. 

kiiehe  (1905) 

Bacillus  thermophilus  alcha. 

Hay 

40°  - 70°  C. 

Hay  in  which  spontaneous  combi^i  ns  had  occurred. 

Anitschkow  (1906) 

59®  - 60®  C. 

Only  occasionally  found  thermophiles  in  alimentary  tract 

(1906) 

Bacillus  thermophilus  alpha 
Bacillus  thormophilus  beta 
Bacillus  thermophilus  gamma 
Eacillus  thermophilus  delta 

Setfage 

52°  - 60°  C. 
optimum 

Ato'.  roc  says  Bardou's  work  on  the  chenical  reactions  of  thermophiles  is  fundamental 
and  far  surpassed  in  detail  *nd  accuracy  other  work  that  had  been  done.  Three  of 
organisms  described  were  denjtrifiers;  all  of  them  were  strongly  proteolytic. 

Blau  (1906) 

B*  cylindricus 
B.  robustus 
B*  tostus 
B.  calidus 

Soil 

to®  c.  cptimuo 

Thermal  death  point  100°  C.  fofl'  20  hours 
Thermal  death  point  100°  C.  for  7 l/2  - 8 hours. 

Extremely  resistant  spores.  Thermal  death  point  100°  C.  for  19-20  hours 
Thermal  death  point  100°  C.  for  3 hours 

Brazolla  (1906) 

Regarded  therr.opb.il  ic  i cleric  cf  s. unit  ary  significance  in  water. 

Falcior.i  (1907) 

B*  thermophilus  I 
B-  thermophilus  II 

Hot  springs 

60°  C.  optimum 

Concluded  tha*  hot  springs  for  vorable  medi . for  thermophilic  bacteria 

Miehe  (1907) 

E.  calfactor 

Hay 

50®  - 60°  C. 
optimum 

Claimed  t > i : : organism  was  responsible  for  he  ti ng  c i h^.  <in/o r tho'th ermo phil e . 

(Maximum  above  coagulation  point  of  protei;^  60°  - 70"  C.) 

Tirelli  (1907) 

Four  rods;  four  cocci;  two 
thread  forms 

Drinking  water 

55°  - 65®  0. 
optimum 

Believed  temperature  relations  due  to  particular  chemical  nature  of  protoplasm  rather 
than  to  their  adjustment  to  the  circumstances  of  their  environment. 

SchOtse  (1908) 

Many  forms  of  B.  cal  factor 
(!Jiehe)  type 

Moist  clover  hay 

Believed  that  thermophiles  grew  better  under  aerobic  conditions  et  high  temperature 
and  at  lower  temperature  they  grew  better  under  anaerobic  conditions 

Jtger  (1909) 

Discussed  possibility  of  spontaneous  combustion  of  different  organic  materials  by 
thermophilic  bacteria. 

Ambroz  (.1910) 

A feomplete  review  of  literature  on  thermophiles  up  to  1910. 

de  Kruyff  (1910) 

fen  rod  forms 

Soil,  water,  air  in  tro- 
pics 

55°  - 6 5°  C. 

Claimed  thermophiles  were  very  abundant  in  tropical  climate.  . 

Proved  thermophiles  were  very  important  in  biological  changes  in  nature. 

. • •• 

B . thermophilus  Vransensis 
B*  thermophilus  Jivioni 
B.  thermophilus  Losanitche 

Hot  springs 

55“  - 60"  0. 

optimum 

43°  - 45°  c. 

optimum 

72°  - 73°  c. 

optimum 

Koch  * Hoffman  (1911) 

Two  spore-forming  bacilli 
One  spore-forming  bacterium 
One  thread  form 

Soil 

52°  c. 

Concluded  that  the  nature  of  the  media  had  a great  influence  on  the  temperature 
response  of  these  organisms,  6ince  they  grew  in  soil  at  a temperature  of  28°  - 30°  C. 
and  would  not  grow  on  artificial  media  at  this  temperature. 

Barlow  (1912) 

Canned  corn 

Aii  organism  said  to  be  responsible  for  spoilage  of  canned  corn 

Kroulik  (1912) 

Tnought  bacteria  end  actinomyces  forms  which  decompose  cellulose  »t  60°  - 65°  C.  are 
widely  distributed  in  nature  and  occur  specially  where  cellulose  is  naturally  decomposed 

Hoick  (1912) 

Review  of  literature 

Ambroz  (1913) 

Denitrobac  erium  thermophilum 

Soil 

2V  C.  elo 
minimum  tem- 
perature 

Called  a true  thermophile  because  it  would  not  grow  at  room  temperature  nor  at  37°  C. 
Ambroz  thinks  the  importance  of  thermophilic  bacteria  in  the  cycles  cf  naturo  can  not 
be  overestimated  since  they  play  such  an  important  role  in  metabolic  processes. 

Negre  (1913) 

numerous  for..s 

Sand  of  Sahara 

Pringsheim  ^ 1913) 

Studied  decomposition  of  cellulose  by  thermophilic  bacteria 

Ko seovric  z (1912-13) 

Studied  thorr.  ophilic  bacteria  in  sugar  juices 

Eergey  (1919) 

Fine  different  bacteria 

DU6t,  soil,  etc. 

60°  - 00°  C. 
maximum 

Suggested  separating  into  two  groups,  the  true  thermo pb.il as  nd  the  : : cultati*e 
thermo phil 66 . 

Cheney  1919) 

Organisms  like  I,  IV7  , and  VI 
of  Rabinowitech 

Canned  foods 

55°“  C. 

- - l o 

supposing  that  nearly  all  cans  containing  them  become  spoiled  and  so  are  automatically 
eliminated  before  reaching  the  market. 

S'  '£ chke  (1919) 

Streptococcus  lacticus  therro- 
philus 

ilk 

roinBirl  (1919) 

: 

B«  aerothernophil ue 
' • thormoal  ; i lontophilus 

Canned  foods 

• 

55°  C.  optimum 
5 5°  c.  optiriun 

Donk  (1920) 

• stearcthernophilus 

Canned  com;  string  leans; 

cc  rn  on  c©i 

50°  C.  opt imun 

ha<  .o  c.for  75  minutes 

C~° 

- s • 1 i • ■ teriw  • Found  th iifcmtfrP 

- .10I4.44- — - 

"obligate  thermophiles” 

Grie, r-Sirit r.  (1920) 

Spore-bearing  rod 

Fermenting  tan-bark 

GO0  C.  optimum 

live  at  80°  c.  in  the  er:  e tin  - rfc  stacks. 

3 

Miquel  (1888)  attributed  the  ability  of  Bacillus 

; 

thermophilus  to  live  and  thrive  at  a temperature  of  70®  C.  and 
above  to  the  particular  character  of  its  protoplasm.  He 
thought,  since  it  grew  at  a temperature  at  which  egg  albumin 
and  blood  serum  were  rapidly  coagulated,  that  this  bacterium 
possessed  a protoplasmic  structure  different  from  that  of  ani- 
mals and  higher  plants. 

Globig  (1888)  did  not  attribute  the  ability  of  the 
bacteria  which  he  isolated  from  soil,  to  grow  at  a temperature 
of  50°  - 70°  0.,  to  any  peculiar  characteristic.  He  thought, 
because  these  organisms  were  found  in  the  superficial  layers 
of  the  earth,  that  the  rays  of  the  sun  furnished  the  heat 
necessary  for  their  growth* 

Fldgge  (1894)  was  probably  the  first  to  report  ther- 
mal death  point  determinations  for  thermophilic  bacteria.  The 
spores  of  the  different  thermophiles  isolated  from  so-called 
sterile  milk  were  found  to  withstand  100°  0.  for  different 
periods  of  time  varying  from  3/4  to  5 hours. 

No  explanation  was  offered  by  Karlinski  (1895)  for  the 
significance  of  the  two  thermophilic  bacteria  isolated  from 
hot  springs.  He  considered  it  remarkable  that  while  the  cul- 
tures of  Bacterium  Ludwigi  lost  their  vitality  at  a temperature 
below  50®  0.  and  were  killed  at  room  temperature,  the  original 
organisms  endured  transportation  in  the  original  water  and  a 


.. 


, 


. 

, • 

. 

‘ 

k 

- 

t 

. 

- 

« 

• 

4 

lowering  of  the  temperature  to  20°  C.  for  several  days*  The 
spores  of  Bacillus  Illidzensis  capsulatus  were  found  to  with- 
stand flowing  steam  at  100°  0.  for  4 minutes* 

Rabinowitsoh  (1895)  found  that  the  spores  of  eight 
thermophilic  Bacteria  isolated  from  various  sources  survived 
steam  at  100®  C*  for  from  5 to  6 hours.  However,  in  spite  of 
this  remarkable  resistance  to  heat,  she  concluded  that  the 
ability  of  the  so-called  thermophilic  bacteria  to  grow  at  such 
high  temperatures  was  a property  of  adaptation  to  environment* 
She  thought  these  organisms  were  mostly  aerobic  but  that  in  the 
absence  of  oxygen  they  would  grow  at  ordinary  temperatures* 

That  thermophilic  bacteria  might  find  conditions 
favorable  for  growth  in  the  temperatures  developed  during  vari- 
ous fermentations  in  nature,  such  as  the  fermentation  of  manure, 
ensilage,  said  moist  hay,  was  suggested  by  Maci’adyen  and 
Blaxall  (1896)* 

The  spores  of  the  oladothrix  form  described  by  Kedzoir 
(1896)  were  found  to  be  very  resistant  to  heat  and  to  disin- 
fectants* They  were  not  killed  by  flowing  steam  at  100®  C*  in 
less  than  3 l/2  to  4 1/2  hours. 

Laxa  (1898)  reported  the  spores  of  Clostridium 
gelatinosum  to  be  very  resistant  to  both  dry  and  moist  heat* 

They  were  not  killed  by  exposure  to  dry  heat  at  150®  C*  for  15 
minutes  nor  by  exposure  to  moist  heat  at  100®  C*  for  75  minutes. 


■ 


* A 

r 

• 

» 

r 

V 

* 

t 

■ 

. 


• - - 


5 

In  the  same  year  Oprescu  (1898)  concluded  from  his 
work  on  thermophilic  bacteria  that  their  biological  properties 
showed  wide  variations  in  relation  to  temperature,  these  vari- 
ations depending  upon  the  media  used.  This  was  the  first  refer- 
ence made  in  the  literature  to  the  fact  that  the  type  of  media 
used  governed  the  temperature  for  the  growth  of  thermophiles. 
About  the  same  time,  Sehillinger  (1898)  concluded 
from  the  results  of  his  experiments  that  in  spite  of  the  fact 
that  these  bacteria  grew  at  such  a high  temperature,  this  temp- 
erature could  not  be  called  the  optimum.  He  also  thought  they 
were  improperly  called  thermophilic  and  that  the  name  thermo- 
tolerant  should  be  applied  to  them. 

The  next  year  Michaelis  (1899),  in  opposition  to 
Sehillinger,  stated  that  the  term  thermophilic  should  be  retain- 
ed and  that  the  high  temperature  for  the  growth  of  the  organisms 
studied  by  him  was  in  all  cases  the  optimum  temperature. 

Mile,  Tsiklinsky*  (1899)  encountered  two  forms  in  soil 

* There  seems  to  be  some  confusion  in  the  literature  with  regard 
to  the  spelling  of  this  investigator *s  name.  The  two  papers 
published  in  the  Annals  1* Institute  Pasteur  bear  the  name  with 
two  different  spellings.  The  names  of  some  of  the  other  invest- 
igators are  also  found  with  two  different  spellings.  This  ac- 
counts for  a few  mistakes  in  the  names  of  authors  in  another 
publication  by  the  present  authors  on  this  subject, 

which  she  called  Ihermoaotinomyees  vulgaris  and  Thermomyces 
lanuginosus.  The  spores  of  the  first  of  these  were  not  killed 
after  20  minutes  at  100°  0.  in  an  autoclave.  The  spores  of  the 


, 

' 


. 


- 

. 


. 


. 


! 


* 

. 


. 


6 

second  one  were  killed  in  1 minute  at  100°  C.  but  withstood 
dry  heat  at  80°  C.  for  3 hours. 

The  most  extensive  work  on  the  thermal  death  points  of 
thermophilic  bacteria  up  to  this  time,  was  done  by  Sames  (1900). 
He  found  that  the  temperature  at  which  the  spores  were  formed 
influenced  their  resistance  to  flowing  steam  at  100°  C.  The 
period  of  time  for  which  the  spores  of  the  eight  Bacilli  de- 
scribed by  him  resisted  steam  at  100°  C.  ranged  from  1 minute 
to  120  minutes.  The  nature  of  the  medium  used  was  also  found, 
as  in  the  work  by  Oprescu,  to  influence  the  temperature  for  the 
growth  of  these  organisms;  separating  them  into  thermophiles  and 
thermo-tolerants,  although  the  line  between  them  was  not  close- 
ly drawn. 

In  an  account  describing  a Micrococcus  found  in  pas- 
teurized milk,  Russell  and  Hastings  (1902)  stated  that  this 
organism  was  able  to  resist  the  action  of  heat  at  76°  C.  for  10 
minutes.  This  fact  seemed  to  indicate  that  the  protoplasm  of 
the  organism  was  markedly  different  from  that  of  most  bacterial 
protoplasm  and  suggested  a "degree  of  tolerance  toward  heat  that 
was  somewhat  analogous  to  that  possessed  by  the  thermophilic 
organisms  in  their  capacity  to  develop  at  abnormal  temperatures". 

Schar dinger  (1903)  studied  one  group  of  bacteria  in 
foods  and  milk  which  grew  between  room  temperature  and  55°  C. 

The  spores  of  two  forms  in  this  group  withstood  an  exposure  to 
boiling  water  for  2 hours,  while  the  spores  of  two  other  forms 


* 


* 


I 


< 


; 


* 

. 


. 

. 

7 


were  killed  in  1 hour  by  a like  exposure. 

In  the  same  year  Setohell  (1903)  carried  out  an  in- 
vestigation of  the  life  of  hot  springs.  He  found  that  bacterial 
forms  endure  the  highest  temperature  observed  for  living  organ- 
isms, being  abundant  at  70°  - 71®  C.,  and  found  in  some  consid- 
erable quantity  at  82°  G.  and  at  89®  C.  In  trying  to  explain 
this  phenomenon,  Setohell  stated  that  there  was  nothing  to  in- 
dicate that  the  protoplasm  of  these  thermophilic  bacteria  con- 
tained so  little  water  as  to  render  it  incoagulable  by  the  high 
temperatures  which  they  endure,  Ho  him  it  seemed  rather  that 
there  might  be  some  important  difference  in  the  essential  pro- 
teids  of  the  mixture  or  in  the  nature  of  the  constitution  of  the 
substance  which  rendered  it  less  coagulable. 

Again  in  1903  Mile,  Isiklinsky  contributed  to  our 
knowledge  of  thermophilic  bacteria  by  an  extensive  report  of 
these  organisms  isolated  from  human  feces.  She  found  that  the 
spores  of  four  Bacilli,  isolated  from  infant  feces,  resisted 
heating  for  5 minutes  at  100®  C.  in  an  autoclave.  As  a result 
of  her  researches  she  concluded  that  thermophiles  were  merely 
variations  of  common  non-thermophilic  organisms,  having  adapted 
themselves  slowly  to  high  temperatures.  She  thought  that  the 
length  of  time  during  which  they  had  adapted  themselves  to  high 
temperatures  made  them  either  facultative  thermophiles  or  ob- 
ligate thermophiles.  She  explained  their  constant  presence  in 
feces  on  the  basis  of  their  wide  distribution  in  nature  and  their 


. 


— 

- 

- 


r - 


•* 

- 

- 

. 

* 

* 

. 

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, 

* * 


- 

■■ 

. 


8 

great  resistance.  Her  results  seemed  to  show  that  all  obligate 
thermophilic  bacteria  were  obligate  aerobes  and  that  the  faculta- 
tive thermophiles  were  facultative  aerobes.  In  this  respect  her 
results  agreed  with  those  of  Rabinowitech, 

Bruini  (1905)  offered  two  possible  explanations  for 
the  existence  of  thermophilic  bacteria  in  the  intestinal  tract. 
Perhaps  they  were  merely  transitional  forms  of  ordinary  bacteria 
from  air  and  foods  and  had  no  part  in  the  fermentation  and  putre- 
faction in  the  intestines,  or  perhaps  thermophiles  found  the 
proper  growth  conditions  in  symbiosis  with  other  organisms  so 
that  they  could  grow  at  temperatures  below  their  optimum. 

The  thermal  death  points  at  100*  C,  of  the  spores  of 
the  four  thermophilic  bacteria  that  Blau  (1906)  isolated  from 
soil  were  found  to  vary  from  7 to  20  hours.  Prom  the  results  of 
his  work,  Blau  reached  the  conclusion  that  varying  the  kind  and 
concentration  of  the  media  changed  to  some  extent  both  the 
morphological  and  physiological  characteristics  of  these  organ- 
isms, 

During  investigations  on  the  spontaneous  heating  of 
hay,  Miehe  (1907)  compared  thermophilic  bacteria  with  ordinary 
bacteria,  and  concluded  there  were  many  transitional  stages  be- 
tween the  two  types,  the  main  groups  being  distinguished  by  their 
minimum  temperatures.  Organisms  that  did  not  grow  at  25°  C.  were 
thermophiles  according  to  Miehe *s  classification;  these  he  again 
divided  into  (1)  orthothermophiles  with  the  maximum  temperature 


, 


. 


> 

• 5 1 ;i  H 


- 


r 


- 


» 


.. 

♦ 

. 

1 


9 


above  the  coagulation  for  albumin  (60°  - 67®  C.)  and  (2)  thermo- 
tolerants  with  a maximum  temperature  of  50°  - 55®  C.f  but  which 
also  grow  well  at  ordinary  temperatures*  The  organisms  which 
grew  best  at  higher  temperatures  but  also  grew,  though  feebly, 
at  lower  temperatures,  he  termed  psychro tolerant* 

In  accordance  with  the  findings  of  Miquel,  Russell  and 
Hastings,  and  Setchell,  Tirelli  (1907),  from  the  results  of  his 
work,  concluded  that  the  ability  of  thermophiles  to  withstand 
such  high  temperatures  must  be  attributed  to  the  particular 
nature  of  their  protoplasm,  rather  than  to  their  adjustment  to 
the  circumstances  of  environment* 

In  work  on  thermophilic  bacteria  in  the  tropics 
de  Kruyff  (1910)  tested  the  resistance  of  the  spores  of  ten  forms 
isolated  from  soil  and  found  that  they  were  killed  in  boiling 
water  in  4 1/2  to  8 hours*  He  also  found  that  thermophilic 
bacteria  grew  better  aerobically  at  the  higher  temperatures  and 
anaerobically  at  the  lower  temperatures*  The  same  results  were 
obtained  by  Rabinowitsch  and  Tsiklinsky. 

Ambroz  (1910)  claimed  that  if  the  high  temperatures  at 
which  thermophiles  would  grow  was  not  the  optimum,  as  stated  by 
Schillinger,  then  thermophilic  bacteria  should  grow  better  at 
ordinary  temperatures  than  they  do  at  the  high  temperatures. 

This  is  not  the  case* 

The  conclusions  drawn  by  Oprescu,  Sames,  and  Blau  were 
confirmed  by  Koch  and  Hoffman  (1911).  The  organism  which  they 
isolated  from  soil  would  grow  on  artificial  media  at  52®  C.,  but 


r 


, 

f 

, 

r 


, 


10 

not  at  25®  - 28°  C.;  in  soil  they  would  grow  at  28®  - SO®  C.  but 
not  so  well  as  at  52®  C.  i'rom  these  results  they  also  concluded 
that  the  nature  of  the  medium  used  had  a great  influence  on  the 
temperature  demanded  for  the  growth  of  thermophiles.. 

Some  spores  of  the  thermophiles  found  in  soil  by  Krou- 
lik  (1912)  were  quite  resistant  to  heat.  The  spores  of  two  of 
these  organisms  withstood  sterilization  with  flowing  steam  for 
2 hours,  while  those  of  two  others  were  a little  less  resistant. 

Noack  (1912)  emphasized  the  importance  of  the  fact 
that  the  resistant  forms  of  thermophilic  bacteria  would  stand 
many  changes  of  temperature  below  as  well  as  above  their  optimum 
temperature.  He  believed  that  this  was  the  reason  they  can  be 
isolated  from  soil  almost  any  time,  whereas,  except  in  the  trop- 
ics, the  temperature  of  soil  seldom  rises  to  the  optimum  temper- 
ature for  these  organisms.  This  might  also  account  for  their 
occurrence  in  decomposing  organic  matter  in  which  the  temperature, 
due  to  spontaneous  heating  seldom  reaches  the  optimum  temperature 
for  their  growth. 

Although  the  thermophiles  studied  by  Uegre  (1912)  were 
isolated  from  the  sands  of  the  Sahara,  they  were  less  resistant 
than  many  of  the  forms  which  have  been  described.  The  spores  of 
these  forms  resisted  heating  at  100®  C.  for  15  to  20  minutes. 

Bergey  (1919)  divided  thermophilic  bacteria  into  two 
groups,  namely,  the  true  thermophiles,  and  the  facultative  thermo- 
philes. In  his  studies  on  many  organisms  in  these  two  groups  he 
found  that  the  spores  of  true  thermophiles  resisted  a temperature 


. 

♦ 

t 

. 


e 


of  100°  C.  for  periods  of  time  varying  from  5 to  400  minutes, 
while  those  of  the  facultative  thermophiles  were  killed  in  15 
to  60  minutes  at  100°  C.  In  the  light  of  this  it  appeared  pro- 

| 

bable  to  Bergey  that  the  optimum  temperature  for  growth  was 

I 

related  to  the  heat  resisting  powers  of  the  spores.  He  oonsid- 

J 

ered  that  the  phenomenal  oharaoteristic  of  thriving  at  temper- 

. 

atures  above  those  at  which  egg  and  serum  albumin  coagulate,  ex- 
hibited by  thermophilic  bacteria,  might  be  due  to  the  reaction 
of  the  medium  in  which  the  bacteria  were  grown,  or  to  the  miner- 
al content  of  their  own  protoplasm. 

While  investigating  the  use  of  a biorizator  for  pas- 
teurizing milk  Patzschke  (1919)  discovered  an  organism  which  he 
named  Streptococcus  laoticus  thermophilus.  This  organism  resist-  | 
ed  a temperature  of  75®  C.  for  3 minutes  but  was  killed  at  65°  C. 
in  10  seconds. 

The  organism  isolated  by  Bonk  (1920)  from  canned  corn 
was  very  resistant  because  it  was  found  in  corn  that  had  been 
processed  at  118*  G.  for  75  minutes. 

The  experiments  of  Bigelow  and  Estey  (1920)  constitute 
the  first  very  extensive  work  on  the  thermal  death  points  of 
thermophilic  bacteria.  They  described  a new  method  of  determin- 
ing  the  thermal  death  points  of  spores,  at  a temperature  of  100° 

C.  and  above,  under  definite  and  well  controlled  conditions.  The 
longest  length  of  time  that  any  of  the  spores  of  the  organisms 
which  they  studied  were  found  to  resist  the  action  of  heat  under 
the  described  conditions  was  1320  minutes  at  100®  G.  This  is  a 


r 


' 

f 

- 

« 

. 

- 

■ . 

- 

* 


' 


, 

- 

, 


12 


greater  degree  of  resistance  than  has  been  reported  by  any  other 
investigator* 

The  spores  of  an  organism  found  to  cause  the  fermenta- 
tion of  spent  wattle-bark  in  the  corrosion  of  white  lead  by 
Grieg-Smith  (1920)  were  very  difficult  to  destroy*  This  was 
especially  true  when  the  spores  were  contained  in  the  pores  of 
the  bark*  They  lived  after  an  exposure  to  186®  to  205®  C*  for 
2 1/2  hours* 

Although  Buchanan  (1922)  gives  no  authority  for  his 
remarks , he  makes  the  statement  that  thermophilic  bacteria  "are 
so  resistant  that  they  will  withstand  boiling  water  literally 
for  days  before  being  destroyed” • 

Thermal  death  point  determinations  and  temperature 
relations  reported  by  the  investigators  included  in  this  survey 
are  summarized  in  the  following  table* 


* 


■I 


* 


, 


r ■■ 


. 


« 


\ 


1 


< 

. > > 
* 


^ w 

- ' r:  t • t 


Tab^e  II 


Invostiyatcr 

Referer.ee 

Organisms  Described 

Source 

Temperature  for  Growth 

Thermal  Death  Points 

(1)  FlStfe  (1894) 

Ztschr.  f . Hyg*  u.  in- 

Menv  bacteria 

Sterilized  milk 

24°  - 44©  C.  or  27°  - 54°  C • 

Spores  Withstood  3/4  to  5 hours  heating  at  100°  c. 

fectionskrnnkh. , 17,  272 

(not  named) 

( 2)  RaUno«d‘.»ch  (1895) 

Ztschr.  f-  Hyg-,  20,  154. 

Bacillus  thermephilue 

I 

Soil;  snow ; 
various  excreta; 
rrains;  milk. 

34°  - 75°  c. 

Spores  survived  steam  at  100°  C.  for  5 to  6 hours 

" 

II 

Spil;  snow; 
excreta;  -rair.s; 

" 

..ilk . 

III 

Soil;  excreta; 

" 

f rair.s;  milk 

" « 

IV 

Soil;  excreta 

" 

" •• 

V 

Excreta;  grain 

" 

" " 

VI 

Excreta 

" 

" 

VII 

Cow  excreta 

" 

" 

" " 

VIII 

Excreta;  grains 

(3)  Karliaeki  (189  5) 

Hyp.  Rundsbau,  5,,  605. 

Bacillus  Illidzer.sis 

Hot  springs  of 

50°  - 50°  C. 

Spores  withstood  flowing  steam  at  100°  C.  for  4 

Capsulatus 

Illid  e ir.  Bosnia 

minutes. 

( 4)  Ked:oir  (189  6) 

Arch.  f.  Hyg.,  27,  328 

A thermophilic  cladothrix 

River  Spree 

35°  - 65°  C.;  55°  C.  optimum 

Spore6  killed  by  flowing  steam  at  100°  C.  in 

form 

3 l/2  7.  4 1/2  hours. 

Ti)  Laxa  (1899) 

Centralbl.  f.  Eekteriol. 

Clostridium  gelatinosua 

"FQllmasse"  in 

25°  - 58°  C. 

Spores  not  killed  by  exposure  to  dry  heat  at  150° 

Att.  11.,  4,  362 

sugar  manufacture 

C-  for  15  minutes  or  moist  heat  i\  lffitf  C.  for 
75  minutes. 

(6)  T.Uin.ky,  lilH.P.  ( 1899 ) 

Ann.  de  l'lr.st.  Pasteur, 

Thermoactinomyces  vulgaris 

Soil 

48°  to  60°  C.;  57°  C-  optinuir 

Spores  not  killed  after  20  minute  at  100°  C. 

13,  500 

Thermomyces  lanurinosus 

» 

42°  to  60°  C. 

in  an  autoclave. 

54°  to  55°  C.  optimum 

Spores  killed  in  one  minute  at  100°  C.;  with- 
stood dry  heat  at  80°  for  3 hourB. 

(7)  SUMS  (19  0) 

Ztschr.  f.  Hyg.,  33,  313 

Bacillus  I 

Earth 

56c  - 70°  C.  optimum. 

At  750.2  ran.  pressure  spores  resisted  live 

70°  - 74°  C * maximum 

steam  for  3 hours  and  10  minutes.  Spores  were 
formed  at  62°  C . 

Eacillus  II 

Pus  from  mouse  in 

56°  - 70°  C.  optimum 

Sporeo  resisted  steam  at  743.7  mm.  pressure  for 

j ected  with  eorth 

75°  C*  maximum 

2 hours  and  50  minutes.  Spores  were  formed  at 

and  immunized 
a- ainst  tetanus 

56°  - 70°  C.  optimum 

62°  C. 

Bacillus  III 

Vaginal  mucous 

Resistance  of  6pores  formed  at  62°  C.  at  740." 

during  pregnancy 
Raw  milk 

66°  - 70°  C.  maximum 

mm.  pressure  = 25  minutes. 

Bacillus  IV 

50°  - 60°  C.  optimum 
66°  - 70°  C.  ma:<imuni 

Resistance  of  spores  formed  at  56°  C.  to  steam 
at  746.1  mm.  pressure  = 15  minutes. 

Resistance  of  9pores  formed  at  62°  C.  to  steam 

Bacillus  V 

Aqueous  litmus 

56°  - 62°  C.kOptimum 

solution 

63°  - 66°  C.  maximum 

at  747.5  rrn.  pressure  = 210  seconds.  Spores 
formed  at  37°  C * at  750.6  mm.  pressure  * 60 

seconds. 

Bacillus  VI 

Same  as  Eacillus 

66°  - 70°  C.  optimum 

Resistance  of  spores  formed  at  62°  C.  to  steam 

II  - 

66°  - 70°  C.  maximum 

at  746.1  ran.  = 13  minutes.  Spores  formed  at 
37°  C.  at  745.3  tnm.  = 2 minutes. 

Bacillus  VII 

Air 

50°  - 60°  C.  optimum 

Resistance  of  spores  formed  at  56°  C.  to  steam 

66°  - 70°  C.  maximum 

at  749.3  mm.  = 100  ninuces.  Spores  formed  at 
37°  C.  at  7 49  . 3 mm.  = 60  to  70  minutes. 

Bacillus  VIII 

Earth 

50°  - 60°  C.  optimum 

Resistiuioe  of  spores  formed  at  56°  C.  to  steam 

, 

66°  - 70°  C.  maximum 

it  745.  1 mm.  = 120  minutes.  Spores  formed  at 
• resistance  to  steam  at  748.9  mm.  * 60 

-%»  1 Afc 

(8)  Russell  ft  Hastinrs  (19S 

Centralbl.  f.  Bakteriol. 

A micrococcus  form 

Pasteurized 

20°  - 25°  C.  optimum 

Thermal  death  limit — 76°  C-  f r 10  minutes. 

Abt.  II.,  8,  339  . 

milk 

76°  C.  maximum 

(9)  TaikliriBky  (1903) 

Ann.  de  l'In6t.  Pasteur, 

Bacille  Ho . 7 

Infant  feces 

24°  - 57°  c. 

Spores  resisted  heating  for  5 minutes  at  100°  C. 

17,  217. 

Bacille  Mo . 8 

37°  C.  to  60°  C. 

in  an  autoclave. 

Spores  resisted  heating  for  5 minutes  ut  100°  C. 

57°  C«  optimum 

in  an 'autoclave. 

. Eacille  No . 9 

42°  to  45°  C_.  optimuv 
57°  C*  optimum 

Spores  resisted  heating  at  100®  C.  in  an  auto- 
clave for  5 minutes. 

Bacille  No.  10 

" " 

Spores  resisted  hentinr  for  5 minutes  at  100°  C. 

Grew  also  at  20°  C. 

in  an  autoclave. 

(10)  Schardinrer  (1903) 

Ztschr.  f.  Untersuch.  d. 

Group  I No.  1 

Foods  etc. 

Room  temperature  - 

Sporee  withstood  2 hours  exposure  to  boiling 

Nahrungs-u.  Genuss. ittel . 

6,  965. 

" No . 2 

.. 

5 5°  C*;  56°  C.  optimum 

water 

" No . 5 

" 

Spores  killed  within  1 hour  in  boiling  water. 

" No . 6 

Spores  did  not  withstand  exposure  of  1 hour 
in  boiling  water. 

(11)  tlau  (1906) 

Centralbl.  f.  Bakteriol. 

B-  cylindricus 

Soil 

60°  - 70°  C»  optimum 

Thermal  death  point  100°  C.  fc  19  to  20  hours 

Abt.  II.,  15,  97. 

B.  robustus 

" 

55°  - 60°  C.  " 

" " " " " 7 l/2  to  3 hours 

B-  t08tU8 

" 

60°  - 70°  C.  " 

" " " " " 19  to  20  hears 

B*  Calidus 

50°  - 65°  C. 

" " " " " 7 to  8 hours 

(12)  d»  Kruyff  (1910) 

Centralbl.  f.  Bakteriol. 

Bacterium  No.  1 

Soil 

37°  - 70°  C;  60°  C.  optimum 

Spores  killed  in  5 l/2  to  6 hours  in  boiling 

Abt.  II.,  26,  65 

..  ..  2 

45°  - 73°  c;  65°  C. 

water. 

Spores  killed  at  10CP  C . in  6 l/2  to  7 hours 

" •'  3 

" 

38°  - 70°  C;  60°  C-  " 

" " " " " 6 1/2  hours 

" " 4 

" 

43°  - 72°  c;  63°  0-  " 

" " 8 hours 

" " 5 

39°  - 67°  0;  60°  C. 

" " 1/2  hours 

,,  6 

35°  - 73°  C;  65°  0. 

..  ..  ..  ..  ,•  4 1/2  hours 

" " 7 

" 

35°  - 70°  C;  55°  C.  " 

" " " " " 5 l/2  to  6 hours 

..  8 

" 

39°  - 70°  0;  58°  0. 

" " " " " 7 l/2  hours 

" " 9 

33°  - 67°  c;  60  c.  " 

" " hours 

« ..  10 

" 

38°  - 68°  C;  60°  C.  " 

••  ■>  " " •'  5 hours 

(13)  Kreulik  (1912) 

Centralbl.  f.  Bacteriol. 

Bacillus  I 1 

Soil 

60°  C.  optimum 

Spores  withstood  2 hours  sterilization  *ith 

Art.  II.,  36,  339 

flowing  steam 

Bacillus  I 2 

60°  ’ . optimum 

Spores  withstood  sterilitation  with  flowing 
stearii  for  2 hours 

Bacillus  II  1 

" 

30°  - 68°  C. 

55°  - 60°  C.  optimum 

Resistance  of  spores  greater  than  that  of  E*  II  2 

Bacillus  II  2 

" 

Same  as  for  B«  II  1 

Spores  did  not  with6tund  the  action  of  flowing 

Bteam  for  2 hours. 

(14)  H.jr.  (1913) 

Corapt.  renc  . Soc.  de 

Bacillus  3 

Sands  of  the 

70°  C.  max.  50°  C.  o<pt. 

Spores  resisted  heating  at  100°  C.  for  15  to  20 

" 4 

" 6 

Sahara 

minutes. 

Investigator 


R)ial  Death  Points. 


(15)  Bergey  (1919) 


(16)  Patzschke  (1919) 


(17)  Donk  (19  20) 


(18)  Bigelow  4 Esty  (1920) 


(19)  Grieg-Smith  (1921) 


Jo 


lal  Death  Point  at  100°  C.  - 400  minutes 

" " - 300  minutes 


M If 


It  II  II  ft 


II  If 


II  II  II  If 


♦ I II 


H «•  lift 


2t-l  death  point  - 75°  C.  for  3 minutes 

temperature  of  85°  C.  for  10  seconds. 


Jo 


II  If 


II  If  II  ft 


H If  tf  ff 


U ff 


- 200  minutes 

- 180  minutes 

- 120  minutes 

- 60  minutes 

- 5 minutes 

- 5 minutes 
-120  minutes 

- 120  minutes 

- 15-60  minutes 


lied  by  processing  at  118°  C.  for  75 
s 


j0iecessary  to  destroy  a known  suspension  of 
27;  in  medium  of  known  hydrogen-ion  concentra- 
lecreases  as  the  temperature  increases. 

'dro gen-ion  concentration  influences  the 
ecessary  to  destroy  a known  suspension  of 
at  a given  temperature, 
itial  concentration  of  spores  influences 
.me  necessary  to  sterilize  a medium  of 
hydrogen-ion  concentration  at  a given 
ature . 


not  killed  after  exposure  to  186°  to  205°  C 
l/2  hours. 


Investigator 

Reference 

Organisms  Described 

Source 

Temperature  for  Growth 

Thermal  Death  points. 

(15)  Be r gey  (1919) 

Jour.Bact.,  4,  301 

Type  1 . 

Type  2 
Type  3 

Type  4 

Type  5 Var.  a. 
Type  5 Var.  b. 
Type  6 
Type  7 

Type  8 Var.  a. 
Type  8 Var.  b. 
Type  9 

Dust;  contamina- 
ted milk  media 
Dust;  soil 

Dust;  soil;  horse 
manure . 

Same  as  Type  3 

Dust;  g.  pig  fec- 
es, horse  manure 
Dust;  cheese; 
pig  feces. 

Horse  manure 

Same  as  Type  6 

Rabbit's  stomach 

Contamination  on 
agar 

Same  as  Type  3 

50°  - 60°  c.  minimum 
75°  - 80°  C*  maximum 
37°  C.  minimum 
70°  C.  maximum 
37°  - 50J  C.  minimum 
70°  - 75°  C.  maximum 
50°  C . minimum 
75°  - 80°  C.  maximum 
37°  C • minimum 
60°  - 70°  c . maximum 
37°  C.  minimum 
60°  C . maximum 
37°  C . minimum 
60°  C.  maximum 
37°  C.  minimum 
50°  C . maximum 
60°  C • minimum 
70°  C.  maximum 
60°  C . minimum 
70° C.  maximum 
20°  - 37°  C • minimum 
50°  - 60°  c . maximum 

Thermal  Death  Point  at  100°  C.  - 400  minutes 

" " " " " - 300  minutes 

" " " " " - 200  minutes 

" - 130  minutes 

" " " " " - 120  minutes 

" " " " " - 60  minutes 

" " - 5 minutes 

" " " " " - 5 minutes 

" " - 120  minutes 

" " " " " - 120  minutes 

" " " " " - 15-60  minutes 

(18)  Patzschke  (1919) 

Ztschr . f.  Hyg-,  31, 
2 26 

Streptococcus  lacticus 
thermophilus 

Milk 

Thermal  death  point  - 75°  C.  for  3 minutes 
Resisted  temperature  of  85°  C.  for  10  seconds. 

(17)  Donk  (19  20) 

Jour.  Bact.,  5,  373 

B.  stearo thermophilus 

Canned  corn 

45°  - 76°  C- 

Not  killed  by  processing  at  118°  C-  for  75 
minutes 

(18)  Bigelow  1 Esty  (1920) 

Jour.  Infect.  Dis., 
27,  602 

19  thermophilic  microor- 
ganisms 

Time  necessary  to  destroy  a known  suspension  of 
spores  in  medium  of  known  hydrogen-ion  concentra- 
tion decreases  as  the  temperature  increases. 

The  hydrogen-ion  concentration  influences  the 
time  necessary  to  destroy  a known  suspension  of 
spores  at  a given  temperature. 

The  initial  concentration  of  spores  influences 
the  tine  necessary  to  sterilize  a medium  of 
known  hydrogen- ion  concentration  at  a given 
temperature. 

(19)  Grieg-Smith  (1921) 

Proc.  Linnean  Soc.  New 
South  Wales,  46,  Part 
I,  76. 

A rod-shaped  bacterium 

Fermenting 
wattle -bark 

Spores  not  killed  after  exposure  to  186°  to  205°  C. 
for  2 l/2  hours. 

13 


III.  Experimental 
Sources  of  Cultures 

Cultures  53  to  87  inclusive,  used 
in  this  investigation,  were  isolated  from  various  samples  of 
soils  and  hog  and  cow  feces.  Part  of  these  samples  were  used 
in  an  investigation  on  the  distribution  of  Clostridium  botulinum 
in  nature  by  Tanner  and  Back  (1922).  Some  of  the  soils  were  tak- 
en from  fields  that  had  been  manured  recently,  sane  from  fields 
that  had  been  treated  with  commercial  fertilizers,  and  some  from 
fields  that  so  far  as  could  be  determined,  had  received  no  treat- 
ment for  the  last  five  years.  All  of  the  soils  tested  were  found 
to  contain  thermophilic  bacteria.  Cultures  88  and  89  were  iso- 
lated from  "Ever  Fresh  Milk",  a commercial  product.  The  cultures 
1 to  52  were  isolated  from  different  samples  of  water  and  were 
used  in  the  master* s thesis  by  the  author. 

Methods  of  Isolation 

Ten  cubic  centimeter  portions  of 
water  suspensions  of  the  different  soils  and  feces  to  be  tested 
were  inoculated  into  large  test  tubes,  each  containing  20  co.  of 
dextrose  broth.  These  tubes  were  incubated  at  55°  C.  for  24 
hours;  from  these  mixed  cultures,  dextrose  agar  plates  were  poured 
in  the  usual  manner  and  incubated  at  55®  C.  for  24  hours.  By 
using  several  dilutions  for  plating  it  was  possible  to  get  well 
isolated  colonies  and  one  thermophilic  bacterium  was  isolated 
from  each  sample  of  soil  or  feces  tested.  The  two  cultures 
from  "Ever  .Fresh  Milk"  were  isolated  from  different  bottles  of 


14 


this  product  by  plating  in  different  dilutions  on  dextrose  agar, 
The  method  of  isolation  used  for  the  thermophilic  bacteria  from 
water  (cultures  1-52)  was  described  in  the  master's  thesis. 
Methods  of  Study 

Inoculations  into  the  different  media 
used  in  this  work  were  made  either  from  24  hour  agar  slant  cul- 
tures or  24  hour  broth  cultures.  The  Descriptive  Chart  of  the 
Society  of  American  Bacteriologists  indorsed  by  them  in  1920  was 
used  in  the  study  of  these  thermophiles,  A copy  of  this  chart 
is  shown  on  the  next  page.  The  index  number  for  each  culture 
was  determined  under  as  uniform  conditions  as  possible.  Some  of 
the  characteristics  of  the  cultures  1 - 52  as  determined  for 
the  group  numbers  of  these  organisms  were  used  in  the  index 
numbers  of  these  cultures. 

Media  and  Technic 

With  one  or  two  exceptions,  the  media 
used  in  this  study  were  those  recommended  by  the  Committee  on  the 
Descriptive  Chart  in  their  report,  "Methods  of  Pure  Culture 
Study",  presented  to  the  Society  of  American  Bacteriologists 
(1919),  All  media  were  tested  for  sterility  by  incubation  at 
55®  C,  for  12  to  24  hours  before  being  used;  the  cultures  were 
all  grown  at  55®  C,  Due  to  the  rapidity  with  which  thermophiles 
grow  it  was  not  found  necessary  to  incubate  test  cultures  longer 
than  4 days,  except  in  the  case  of  milk  cultures  which  were 
incubated  for  7 to  10  days.  Two  per  cent  agar  was  used  through- 
out this  investigation  because  it  seemed  to  be  more  suitable 


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SPECIAL  TESTS  (e.  g.  PATHOGENICITY) 


15 


for  incubation  at  55®  C#  than  the  1 or  1 l/2  per  cent  agar  as 

» 

ordinarily  used* 

i 

Microscopic  features 

i 

j 

The  microscopic  features  were 

determined  by  use  of  carbol  fuohsin  and  Oram  stains#  ior  the 

) 

staining  of  flagella  the  nev/  method  proposed  by  Plimmer  and 
Paine  (1921)  was  used,  with  certain  modifications,  and  proved 
to  be  a very  quick  and  efficient  method#  Since  it  was  found 
that  the  thermophilic  bacteria  under  observation  grew  very  rap- 
idly at  55®  C.,  8 - 12  hour  agar  slant  cultures  were  used#  Some 
of  the  growth  was  removed  carefully  and  put  into  tubes  contain- 
ing sterile  water  which  had  been  held  at  a temperature  of  55®  G# 
for  about  an  hour#  These  water  suspensions  were  kept  at  55®  G# 
for  30  - 60  minutes  and  then  2 or  3 small  drops  of  the  suspen- 
sions were  placed  on  slides  that  had  been  prepared  according  to 
the  description  of  Plimmer  and  Paine  and  maintained  at  55®  C# 
for  15  - 60  minutes#  It  was  found  that  by  keeping  the  suspen- 
sions and  slides  at  55®  G#,  thus  avoiding  any  change  in  temper- 
ature during  the  preparation  of  the  smears,  that  the  motility 
was  not  interfered  with  and  the  flagella  were  then  easily  demon- 
strated by  staining#  The  smears  were  allowed  to  dry  at  55®  G. 
over  night  and  then  fixed  and  stained  by  the  method  as  described 
by  the  above  mentioned  authors. 

Miscellaneous  Biochemical  Reactions 

Just  one  of  the 

cultures  (iio.  4)  was  used  to  test  its  pathogenicity  for  guinea 


16 

pigs*  This  culture  was  grown  on  blood  agar  slants  for  18  hours, 
the  growth  removed  and  a suspension  of  it  in  physiological  salt 
solution  made*  Guinea  pigs  were  given  injections  of  this  sus- 
pension intraveneously  and  interperitoneally  with  no  effect* 
Bruini  (1905)  is  the  only  investigator  who  attributed  a patho- 
genic effect  to  a thermophile*  A zero  is  used  for  this  charac- 
teristic in  all  of  the  index  numbers  simply  to  indicate  not  that 
it  was  not  pathogenic,  but  that  this  characteristic  was  not 
studied  on  all  of  the  cultures. 

For  the  determination  of  gelatin  liquefaction  the 
provisional  method  was  used*  It  is  designed  to  distinguish 
"true  liquefiers"  (organisms  producing  ecto-enzymes ) from  organ- 
isms that  produce  endo-enzymes  of  proteolytic  action  that  are 
released  from  the  cell  after  death  and  cause  liquefaction  of 
the  gelatin  if  incubated  for  a long  period*  The  cultures  were 
given  a preliminary  cultivation  for  24  to  48  hours  (according 
to  the  rapidity  of  growth)  in  a 1 per  cent  solution  of  gelatin 
at  55°  C*;  then  the  surface  of  gelatin  in  test  tubes  was  inoc- 
ulated and  incubated  at  55°  C*  for  4 days*  By  this  method  all 
the  cultures  except  78,  83,  and  84  were  found  to  be  gelatin 
liquefiers. 

To  test  for  the  production  of  nitrites  and  gas  in 
nitrate  media  both  nitrate  broth  and  nitrate  agar  slants  were 
used. 


f 


. 


4 

* 

r 

- 


« 


. 


' 


> 


: . 


, 


» 


- v: 


17 


Carbohydrate  Reactions 

The  method  of  Baker  (1922)  was 
used,  with  certain  modifications,  to  determine  the  production 
of  acid  and  gas  in  dextrose,  laotose,  and  sucrose  broths.  Brom 
thymol  blue  was  added  to  these  carbohydrate  broths  before  the 
media  was  tubed  and  sterilized.  It  was  found  that  the  addition 
of  15  co.  of  a 0.04  per  cent  alcoholic  solution  of  the  indicator 
to  every  liter  of  carbohydrate  broth  made  a good  concentration 
for  the  detection  of  acid  production,  and  was  not  strong  enough 
to  inhibit  growth. 

J’or  the  determination  of  diastatio  action,  starch  agar 
plates  were  used  and  dot  inoculations  were  made  in  the  center 
on  the  surface  of  the  agar  in  the  plates  instead  of  the  usual 
streak  inoculations. 

Other  Characteristics 

Tests  for  indol  were  made  on 

nutrient  broth  and  Dunham's  peptone  solution  cultures;  Vsfitte's 
peptone  was  used  in  these  media  since  it  seemed  to  be  better 
suited  for  the  production  of  indol  by  bacteria.  Both  the 
nitroso-indol  test  and  Ehrlich's  test  were  used.  Ehrlich's 
reagent  was  prepared  according  to  the  method  described  by  Horton 
and  Sawyer  (1921).  It  was  found  that  this  test  gave  more  satis- 
factory results  when  the  tubes  were  heated  slightly.  All  the 
cultures  studied,  except  Ho.  81,  produced  indol*  In  general 
the  conclusions  of  Horton  and  Sawyer  were  confirmed. 


18 


For  the  determination  of  the  production  of  hydrogen 
sulfide,  nutrient  broth  made  with  Witte's  peptone,  over  which 
a strip  of  lead  acetate  paper  was  suspended  by  means  of  the 
cotton  plug,  was  used*  The  blackening  of  the  paper  indicated 
hydrogen  sulfide  formation*  Streak  cultures  on  ”Bacto  Lead 
Acetate  Agar”  plates  were  also  used*  All  but  cultures  No.  83 
and  84  formed  hydrogen  sulfide* 

Litmus  milk  and  sterile  milk  to  which  brom  cresol 
purple  had  been  added,  were  used  to  determine  the  reactions  of 
the  thermophiles  in  milk.  These  milk  cultures  were  incubated 
7-10  days.  Most  of  the  cultures  grew  well  in  milk;  only  two 
cultures,  N03.  54  and  59  produced  no  apparent  change  in  the  milk. 
Cultures  No.  83  and  84  produced  alkali  with  no  other  apparent 
change.  The  majority  of  the  rest  of  the  cultures  showed  coag- 
ulation and  peptonization  with  an  alkaline  reaction,  some  of 
them  having  shown  a slight  preliminary  acidity.  A few  cultures 
showed  slight  acidity  in  milk  and  a few  showed  distinct  acidity 
with  coagulation  but  no  digestion  of  the  casein. 

Temperature  Relations 

Of  the  89  cultures  used  in  this 

investigation  18  cultures  were  chosen  for  use  in  a more  intensive 
study  of  temperature  relations  and  thermal  death  point  deter- 
minations. The  choice  of  these  18  cultures  was  made  to  include 

organisms  from  as  many  sources  as  possible*  These  cultures  were 
grown  on  agar  slants,  in  gelatin  tubes,  and  on  agar  plates,  at 


, 


4 


.. 

. 

: . ‘ . .> 


, 

4 


' 


19 


the  five  different  temperatures  available  for  incubation  in 
these  laboratories*  The  agar  slants,  5 for  each  culture,  were 
inoculated  by  means  of  a small  wire  loop  from  a 24  hour  broth 
culture  of  the  organism  to  be  tested,  and  these  agar  slants 
incubated  simultaneously  at  the  five  given  temperatures*  The 
gelatin  was  inoculated  by  placing  a loopful  of  24  hour  gelatin 
solution  cultures  on  the  surface  of  gelatin  in  tubes*  Five  tubes 
were  prepared  for  each  organism,  and  these  tubes  incubated  at 
the  five  given  temperatures*  Large  petri  dishes  were  used  for 
the  agar  plate  cultures,  dot  inoculations  being  made  in  the  cen- 
ter of  the  plate  on  the  surfaoe  of  the  hardened  agar.  A small 
wire  loop  was  used  to  make  these  inoculations  from  24  hour  broth 
cultures  of  the  organisms. 

All  of  these  inoculated  media  were  incubated  at  the 
five  given  temperatures  for  24  hours  in  order  to  have  a definite 
period  of  incubation;  a 24  hour  incubation  period  was  used  be- 
cause the  agar  streak  cultures  and  plate  cultures  seemed  to 
reach  their  maximum  growth  within  such  a period  of  incubation* 

The  comparative  amount  of  growth  attained  in  gelatin  and  on  agar 
slants  at  the  different  temperatures  was  judged  as  carefully 
and  as  accurately  as  possible  with  the  naked  eye;  the  diameter 
in  mm*  of  the  giant  colonies  produced  on  the  agar  plates  at  these 

same  temperatures  was  measured. 

The  data  secured  are  given  in  the  following  table 
{Table  III),  i’igures  are  used  to  indicate  the  amount  of  growth, 


- 


■ ' 


* 


, 

. 


. . . , ■ . 


; 


. 

' 


. 

» 


20 


1 indicating  the  greatest  amount  of  growth  at  the  stated  tem- 
peratures, 2 the  next  best,  etc.  This  method  is  used  for  the 
lack  of  a better  one* 


The  method,  proposed  by  Bigelow  and  Estey  (1920),  for 
the  determination  of  thermal  death  points  of  typical  thermophilic 
organisms,  with  a few  modifications,  was  followed  in  this  study. 

Nutrient  agar  slants  were  inoculated  with  pure  cultures 
of  the  organism  to  be  tested  and  grown  at  55°  0.  for  48  hours. 

The  growth  from  two  agar  slants  of  each  culture  was  brought 
into  suspension  by  pouring  10  co.  of  sterile  nutrient  broth  onto 
the  slants  and  emulsifying.  These  suspensions  were  then  trans- 
ferred to  flasks  containing  100  cc.  of  sterile  broth  and  incu- 
bated for  7 days  at  55°  G.  At  the  end  of  the  incubation  period 
the  flasks  were  placed  in  a refrigerator  for  24  to  48  hours, 
when  they  were  heated  to  85°  0.  for  15  minutes  to  kill  all 
vegetative  forms,  cooled  immediately,  and  placed  again  in  the 
refrigerator  to  prevent  the  germination  of  spores.  These  were 
the  stock  suspensions  used  for  the  determination  of  the  thermal 
death  points  of  the  spores.  The  concentration  of  spores  in  the 
suspensions  was  determined  by  plating  in  different  dilutions. 

The  relative  ability  of  the  18  cultures  to  form  spores  under 
these  conditions  is  shown  by  the  counts.  The  number  of  spores 
per  cc.  of  suspension  for  the  18  cultures  varied  from  590  to 


. ■ ' 


. 


. 

. 

* 

. 


„ 


. 


, 

. 


Table  XII 

GROWTH  OF  THERMOPHILES  ON  3 DIFFERENT  MEDIA  AT  5 TEMPERATURES  (CENTIGRADE) 

Agar  Plate 

0 

o 

to 

COtO'tf'^tOtOtOr— ltOtOOiO)O0tOO3tOO3rH 

o 

LO 

m 

HOJIONHHHOJNNHHHHHHHOJ 

o 

m 

i 

o 

03 

03rH.HiHC3  03O3tOrHiHO3rHtOO3tOO3tOtO 

o 

J> 

to 

■^•^OSlO^^’^'^'^^tOtOtOtO^tO  1 1 

0 

o 

to 

1 

0 

m 

03 

^mioin-tfio^^T^in^^Tit^LO^  • 1 

Gelatin 

O 

o 

to 

1 | ^ |03t0t0^'^^lH^^-^rHrH 

0 

m 

m 

t0r-ltOt0r-l03iH0lr-ltOtQtO03  03r—lrHO3O3 

0 

m 

i 

0 

03 

H03HH03HtOH01rHHHtOHN03tOtO 

o 

O' 

to 

03t00303t0t0^^^030303'^t0t0t0^'^ 

o 

o 

to 

1 

o 

m 

03 

to  ^ in  n i iininminm  i i i im 

Agar  Slant 

© 

o 

to 

03  1 t003tOt003t003rHrHrHrH03t0^rH03 

0 

m 

in 

HrlrlHHHHHH03NN03HHrl03H 

0 

m 

a 

0 

03 

t00303t00303t003t0t0t0t0t0t00303t0t0 

0 

O' 

to 

0 

0 

1 

0 

m 

03 

m i tP  i i in  m m i i i nn  i i 

Culture 

Number 

OOiHtOtOOWlOOHWtOO^OOOi 

rHrHrHO3O3tOtOtO'^*'^iniOinintOtO00C0 

' ' . • 


I I 


I I 


1 


I 


I 1 


I 


lilt 


J . . I . J 


r r i [ill 


i i 


M 


73,800,0^0 


21 

The  tubes  used  for  the  determinations  were  hard  glass 
tubes  5 mm.  in  diameter  and  250  mm.  in  length;  they  were  pre- 
pared for  use  by  soaking  over  night  in  weak  hydrochloric  acid 
solution,  rinsing  thoroughly  with  distilled  water,  draining, 
wrapping  in  heavy  brown  paper  in  packages  of  15  each  and  ster- 
ilizing. These  tubes  were  inoculated  with  1 cc.  of  the  sus- 
pensions of  spores,  sealed  off  to  within  40  to  50  mm.  of  the 
surface  of  the  liquid,  and  held  in  the  refrigerator  until  ready 
to  be  heated. 

The  thermal  death  points  of  the  spores  at  100°  C., 

105°  C.,  110°  C.,  115°  C.,  and  120°  C.  were  determined  by  im- 
mersing the  sealed  tubes  in  an  oil  bath  adjusted  to  the  desired 
temperature.  A DeKhotinsky  electric  bath  containing  "Crisco" 
was  used  to  maintain  a constant  temperature,  and  a Wassernan 

test-tube  rack  for  suspending  the  sealed  tubes  in  the  bath. 

Before  immersing  the  sealed  tubes  in  the  oil  bath  the  tempera- 
ture was  increased  one  degree  in  order  to  compensate  for  the  loss 
in  temperature  due  to  the  immersion  of  the  tubes  in  the  oil  and 
30  seconds  were  allowed  for  the  heat  to  reach  the  center  of  the 
tubes  and  for  the  temperature  to  drop  to  that  at  which  it  was 
previously  adjusted,  before  recording  the  time. 

A series  of  tubes  was  exposed  to  the  desired  temper- 
ature for  definite  periods  of  time,  a tube  being  removed  at  the 
end  of  each  period  and  immediately  placed  in  a bath  of  ice  water 


r 


zz 

in  order  to  prevent  further  action  of  the  heat  on  the  spores  of 
the  bacteria.  When  cold,  the  tubes  were  placed  in  the  refrig- 
erator and  held  until  the  sterility  of  the  medium  could  be 
determined. 

Sterility  was  determined  by  inoculating  agar  plates 
with  the  contents  of  the  heated  tubes  and  incubating  for  2 to 
4 days.  In  most  oases,  when  spores  had  survived  the  heating, 

growth  occurred  within  24  hours,  and  in  no  case  did  growth  occur 
after  48  hours. 

The  hydrogen-ion  concentration  of  the  suspensions  was 
determined  at  the  beginning  and  end  of  each  period  of  heating 
by  the  colorimetric  method.  The  color  chart  of  indicators  show- 
ing the  colors  of  Clark  and  Lube*  indicators  in  solutions  of 
known  pH  was  used  to  determine  the  pH  of  the  different 
suspensions. 

Charts  were  prepared  to  show  the  thermal  death  points 
of  these  18  thermophiles  at  the  five  given  temperatures.  These 
follow. 

Table  IV,  following  the  charts,  summarizes  the  work 
of  the  thermal  death  point  determinations  on  these  organisms  and 

also  indicates  the  change  in  pH  of  the  broth  suspensions  due  to 
the  heating. 


* 


4 


, 


' 


r « 


23 


24 


26 


Table  IV. 

Tim©  Required  to  Destroy  the  Spores  of  Thermophiles  at  Stated  Temperatures. 

Time  Required  to  Destroy  and  Changes  in  pH  at 

• 

© 

o 

o 

CM 

r-l 

w 

Q« 

cocooocDcococoyD^QOcoooocoor)cofo 

c^r-i>L*-c<-i>ot-!>oi>aocococ^c^£>j> 

i • i i i i i i i i i i i i i i i i 

C0OC0cOr0(O(0t.0^(0  03OOOQDC0cnt0 

••♦•••••••••  • • • • o O 

OO£>C'-OC'-OC-C-I>C-C0C0Q0t>Ol>£> 

Min. 

ooooomomoifloifloooiooii) 

oOu)ioiowu)>ot>ir)No^ioo3int> 

W ^ H rH  rH  rH  rH  rH  rH  iH  rH  rH  rH  rH  rH  rH  rH  rH 

• 

O 

0 

o 

H 

H 

a 

OOOQOOCOCOOOiiCOCOOOOCOQDcncO 

1 1 1 t 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

OOcOCOcOCOcc-cD'd^OOCOOOOCOCOCOcO 

Min. 

OOOOOOOOOOOOOOOOQO 

lOOOOOOOOOOiOOOOiOOOO 

.ft... 

CMCMCMCMCMCMCMCMCMCMCMCMCMtOCMCMtOtO 

♦ 

o 

o 

o 

H 

H 

w 

p. 

COOCOOOOOOO^COCOOOOCOCQOOcO 

C-OO-£>C>r>J>-L''^i>l>C000C0t>£>t>C> 

1 1 1 t 1 1 i 1 1 1 1 1 1 1 1 1 1 1 

OTOCOCCOCDOcOt^QCOOOOCOCOCOcO 

C-C'-t>t~£>I>C'-t>!>I>l>COCOCDI>-C'-t>I> 

Min. 

^toioiocon^^inioint't-C'toinacR 

• 

o 

o 

lO 

o 

rH 

w 

Ot 

OOtOCOcOQDcO^OCO^COOOOOCOOCOCncO 

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

oo'vDcococx)«o^oco^cocoooococooa«o 

!>[>C^t>l>t'OC^>>C'COCOCOt>t'Cv-f' 

» 

C3 

•H 

2 

lOOOOOOOOOOOOtOOOOOO 

^-HCMtOtQiOtO'tfiOtQCMtOCM^LOcDt-tO 

•OoOOT 

pH 

o<ocococo<o<oootoo>oQoooao  o*o>  cn 

coc^t~i'~kN-t'C^L'>t~i>L'~ooa5aDr~c>f>!> 

f 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 J 

cDcoooy5coto<oy>^QaoDooooocoooco 

t>£>£>£>-C^>£>C,-l>l>>OOo6o5l>l>l>C> 

Min. 

oiooooooooooo  m o o o o o 

HH^mao<ocoa»o>c\)t><ocot>t>o>cMoo 

rH  r-l  pH  rH  CM  rH 

Spores 
per  cc. 

oooooooooooooooooo 

OOJOOOOOOOOOOOOOOOO 

OtOOOOOOOOOOOOOOOOO 

O lOOOf'OlQOOiOO^^O^OrH 
CO  t*<OOCMOCM'^C'C'OQO^,M'^OH 
LO  LOO  CO  rH  rH  CM  O rH  rH  <0  LO 

. . . It  «i 

to  LO  00  to  rH 

c- 

Culture 
Numbe  r . 

r-IOO>rHtOiO£>OtOC'-.HCM  IQ  0>  ^ t>  O O* 
rHrHCMCMtOtOtOH<^iOiOlOlO'OtOOOCO 

i i i I i i i I i i r i t I l ! i t 


1 I I I I I I I ! I 1 I I 1 I I I I 


1 I I 


I I I 


I [ I | I I I I 1 I 


28 


Index  Numbers 

Table  V.  includes  the  source  and  "Index 
dumber"  for  each  organism  studied.  On  page  29  is  found  a key 
to  be  used  to  determine  what  characteristics  of  these  thermo- 
philes  are  indicated  by  the  figures  in  the  given  index  numbers. 


Another  table  (Table  YI ) shows  the  separation  of  the 
thermophiles  studied  into  classes  according  to  the  index  numbers. 


. 


■ 


29 


Index  No.* 

BRIEF  CHARACTERIZATION 

As  each  of  the  following  characteristics  is  determined,  indicate  in  proper  marginal  square  by 
means  of  figure,  as  designated  below: 


C/5 

O 

o 

*S.  w 

o <v 

a 3 

Form:  1.  streptococci;  2,  diplococci;  3.  micrococci;  4,  sarcinac;  5.  rods; 

6.  commas;  7.  spirals;  8.  branched  rods;  9,  filamentous 

Spores:  1.  central;  2.  polar;  3,  absent 

£ 

V-*  <0 

a* 

Flagella:  1.  peritrichic;  2.  polar;  3.  absent 

2 

w 

H 

o 

Gram  stain:  1.  positive;  2,  negative 

3-d 

S .p  a 
S § .2 
S J8  O 

Pathogenicity,  etc.:  1,  for  man;  2,  for  animals;  3.  for  plants;  4,  parasitic 

but  nqt  pathogenic;  5,  saprophytic;  6.  autotrophic 

Relation  to  oxygen:  1,  s‘rict  aerol  e;  2 facultative  anaerobe;  3,  strict  anaerobe 

w 

o 

Gelatin  liquefaction:  1,  positive;  2.  negative 

8|  S 

JSadi 

S® 

In  nitrate  media:  1,  nitrite  and  gas;  2.  nitrite  but  no  gas;  3,  neither  nitrite 

nor  gas 

>! 

Chromogenesis:  1,  flourescent;  2.  violet;  3.  blue;  4.  green;  5.  yellow; 

6,  orange;  7,  red;  8.  brown;  9,  pink;  0,  none 

2 

a 

<D 

Ctf  CO 

Diastatic  action:  1,  positive;  2,  negative 

U B 

From  dextrose:  1,  acid  and  gas;  2,  acid  without  gas;  3.  no  acid 

& 

O cJ 
US  0/ 

53  « 
o 

From  lactose:  1.  acid  and  gas;  2,  acid  without  gas;  3.  no  acid 

From  sucrose:  1,  acid  and  gas;  2,  acid  without  gas;  3,  no  acid 

<D 

Diameter:  1.  under  O.oM;  2.  between  O.oM  and  IM;  3.  over  1M 

CO 

o 

C— 1 

> 

+3  CO 
ctf 

Length:  1.  less  than  2 diameters;  2,  more  than  2 diameters 

<l)  " 

bO  vJ 
a> 

> 

Chains  (1  or  more  cells):  1,  present;  2,  absent 

C/3 

M 

Capsules:  1,  present;  2,  absent 

2 

w 

CO 

a> 

u 

Shape:  I,  round;  2,  oval  to  cylindrical 

r* 

o 

Cfl 

Diameter:  1,  less  than  diameter  of  rod;  2,  greater  than  diameter  of  rod 

W 

o 

<D 

O 

Abundance:  1,  abundant;  2,  moderate;  3,  slight;  4.  absent 

to 

w 

53 

no 

< 

Lustre:  1,  glistening;  2.  dull 

§ 

a> 

u 

2 

Surface:  1.  smooth;  2,  contoured;  3.  rugose 

<i 

Q 

0) 

b 

Agar  colonies:  1,  panctiform;  2,  round  (over  1 mm.  diameter);  3.  rhizoid; 

4,  filamentous;  5,  curled 

\ O 
u 
w 
1/1 

cU 

3 

Gelatin  colonies:  1,  punctiform;  2,  round  (over  1 mm.);  3.  irregular;  4.  fila- 
mentous 

3 

Acid:  1,  sufficient  for  curdling;  2.  insufficient  for  curdling;  3.  no  acid 

3 

Rennet  curd:  1,  present;  2.  absent 

Peptonization:  1,  present;  2.  absent 

•Recording  the  "Index  Number”  here  is  optional:  but  its  use  will  be  found  convenient  if  the 
charts  are  to  be  filed  according  to  the  salient  characteristics  of  the  organisms.  The  Index 
Number  consists  of  the  first  thirteen  figures  from  the  margin  (primary  characteristics)  copied 
down  in  the  order  of  their  occurrence  in  the  margin,  placing  a dash  wherever  a heavy  rule 
occurs  in  the  margin.  Thus.  B.  coli  belongs  to  the  group  5312— 41220-i 111. 


. • 


' 

■ 


Table  V. 

Culture 

Source 

. Index  Number. 

Number. 

1 

Dug  well,  Champaign 

5111-01120-1232 

2 

Raw  water,  Hamilton 

5111-01120-1232 

3 

Deep  well.  Sears  Roebuck 

5211-01120-1232 

4 

Boneyard" , Urbana 

5111-01120-1232 

5 

18  ft.  dug  well,  Odin 

5211-01120-1232 

6 

Raw  water,  Pontiac 

5111-01120-1232 

7 

City  supply,  Joliet 

5211-01120-1232 

3 

I.C.  Railroad 

5211-01120-1232 

9 

Raw  water,  Alton 

5111-01120-1232 

10 

Raw  water,  Kankakee 

5111-01120-1232 

11 

Raw  water,  Decatur 

5211-01120-1232 

12 

20  ft.  dug  well,  Newman 

5211-01120-1232 

13 

Raw  water,  Q,uincy 

5111-01120-1232 

14 

City  supply,  Lake  Forrest 

5111-01120-1232 

15 

Raw  water,  Moline 

5111-01120-1232 

16 

30  ft.  dug  well,  Macomb 

5111-01120-1232 

17 

City  supply,  Waukegan 

5111-01120-1232 

13 

City  supply.  Oak  Park 

5111-01120-1232 

19 

145  ft.  driven  well,  Dickson 

5111-01120-1232 

20 

200  ft.  drilled  well,  " 

5111-01120-1232 

21 

25  ft.  wdll,  Chicago 

5211-01120-1232 

22 

Tap  water,  DeKalb 

5211-01120-1232 

23 

75ft.  drilled  well,  Peoria 

5211-01120-1232 

24 

22  ft.  dug  well,  Carmi 

5111-01120-1232 

25 

40  ft.  dug  well,  Newman 

5211-01120-1233 

26 

Raw  water,  Danville 

5111-01120-1232 

27 

Well,  C.&A.  Railroad,  Bloomington 

5211-01120-1232 

28 

Raw  water,  Herrin 

5111-01120-1232 

29 

Spring,  Bloomington 

5121-01120-1233 

30 

19  ft.  dug  well,  Waverly 

5211-01120-1232 

31 

35  ft.  dug  well,  Cardiff 

5111-01120-1232 

32 

Well,  Champaign 

5111-01120-1232 

33 

City  supply,  Belleville 

5111-01120-1232 

34 

Well  water,  Streater 

5111-01120-1232 

35 

Well,  Bloomington 

5111-01120-1232 

36 

Well,  Bloomington 

5111-01120-1232 

37 

Well,  Bloomington 

5111-01120-1232 

38 

Raw  water,  Carlinville 

5111-01120-1232 

39 

City  supply,  " 

5111-01120-1232 

40 

City  supply,  Quincy 

5111-01120-1232 

41 

City  supply,  Alton 

5111-01120-1232 

43 

Spring,  Harris town 

5111-01120-1232 

44 

Galesburg,  well 

5111-01120-1232 

45 

Mississippi  river,  Moline 

5111-01120-1232 

46 

City  supply,  Moline 

5111-01120-1232 

47 

Raw  water.  Rock  Island 

5111-01120-1232 

48 

30  ft.  dug  well.  Forest 

5111-01120-1232 

49 

20  ft.  dug  well,  Matoon 

5111-01120-1232 

50 

Raw  water,  East  St.  Louis 

5111-01120-1252 

* 


. I 


. D 

* . I 


t. 


t 


t 


t 


"N 


c 


( 


0 


Table  V. 
(Continued) 

Culture 

Number. 

Source 

Index  Number. 

51 

City  supply,  East  St.  Louis 

5111-01120-1232 

52 

30  ft.  dug  well,  Winchester 

5111-01120-1232 

53 

Hog  feces 

5111-02120-1232 

54 

Hog  feces 

5111-02120-1232 

55 

Hog  feces 

5111-01120-1232 

56 

Soil  from  old  hog  lot 

5111-01120-1232 

57 

Soil  from  dry  hog  lot 

5111-02120-1232 

58 

Soil  from  vegetable  garden 

5111-02130-2333 

59 

Soil  from  city  garden 

5111-02120-1232 

60 

So il  from  garden 

5111-02120-1232 

61 

Soil  from  corn  field.  Not  manured  with- 
in last  5 years. 

5111-02120-1232 

62 

Soil  from  corn  field.  Manured 

5111-02120-1232 

63 

Soil  from  corn  field.  Manured 

5111-02120-1232 

64 

Soil  from  corn  field.  Not  manured  with- 
in last  5 years. 

5111-02120-1232 

65 

Cow  feces. 

5111-02120-1232 

66 

Cow  feces. 

5111-02120-1232 

67 

Cow  feces. 

5111-02120-1232 

68 

Soil  from  experimental  plot. 

5111-02120-1232 

69 

Soil  from  vegetable  garden. 

5111-02120-1232 

70 

Garden  soil. 

5111-02120-1232 

71 

Garden  soil. 

5111-02120-1232 

72 

Soil  from  city  garden. 

5111-02120-1232 

73 

Soil  from  garden  (new  ground) . 

5111-02120-1232 

74 

Garden  soil. 

5111-02120-1232 

75 

tl  tt 

5111-02120-1232 

76 

tt  It 

5111-02120-1232 

77 

Soil  from  corn  field. 

5111-02120-1232 

78 

Soil  from  corn  patch.  Manured. 

5111-02220-1232 

79 

Soil  from  land  next  to  railroad. Sown 
to  oats. 

5111-02120-1232 

80 

Soil  from  pea  field,  Northern  Illinois 

5111-01130-2333 

81 

Soil  from  corn  breeding  plots.  Land 
formerly  in  cattle  feeding  lots. 

5212-01130-2333 

82 

Soil  from  oat  field. 

5111-02120-1232 

83 

Soil  from  corn  field. 

5111-02230-1222 

84 

Soil  from  oat  field. 

5111-02230-2222 

85 

Soil  from  corn  field. 

5121-02120-1232 

86 

Soil  from  pea  fie Id, Northern  Illinois 

5111-01120-1232 

87 

n h tt  it  it  t» 

5111-02120-1232 

88 

"Ever  Fresh  Milltw 

5111-01120-1232 

89 

tt  tt  It 

1 

5111-02120-1232 

) 


0 


( 1 


Table  VI. 

Class 

Index  Number 

Culture  Numbers 

Number  of 
Cultures  in 
each  Class. 

I 

5111-01120-1232 

1,2,4,6,9,10,13-20,24, 

26,28,31-41,43-52,55, 

56,86,88. 

42 

II 

5211-01120-1232 

3,5,7,8,11,12,21-23, 

27,30. 

11 

III 

5211-01120-1233 

25. 

1 

IV 

5121-01120-1233 

29. 

1 

V 

5111-02120-1252 

53,54,57,59-77,79,82, 

87,89. 

26 

VI 

5111-02130-2333 

58. 

1 

VII 

5111-02220-1232 

78. 

1 

VIII 

5111-01130-2333 

80. 

1 

IX 

5212-01130-2333 

81. 

1 

X 

5111-02230-1222 

83. 

1 

XI 

5111-02230-2222 

00 

• 

1 

XII 

5121-02120-1232 

85. 

1 

0 


3 


^ ^ t 


I 


30 

The  organisms  in  Class  I differ  from  those  in  Class  II 
only  in  the  location  of  the  spore.  Those  in  Class  I had  central 
spores  and  those  in  Class  II  had  polar  spores.  This  differentia- 
tion is  made  on  a character  of  minor  importance  in  classification 
and  analysis  of  bacterial  groups.  Consequently,  the  bacteria 
in  Classes  I and  II  may  be  regarded  as  belonging  to  the  same 
olass  when  merely  spore  formation  itself  is  considered,  Dushnell 
(1922)  has  called  attention  to  the  difficulties  arising  when 
only  one  characteristic  such  as  location  of  the  spore  in  the  rod 
is  considered.  The  two  classes  (I  and  II)  are  left  separated  in 
this  summary  since  the  separation  is  called  for  on  the  latest 
Descriptive  Chart  and  because  some  of  the  recent  reports  on 
anaerobic  spore  formers  indicates  this  to  be  a fairly  constant 
characteristic,  hall  (1922)  stated  that  three  distinct  groups 
of  anaerobic  spore  formers  could  be  differentiated  on  location 
of  the  spore,— 

I,  bacteria  with  central  spores  which  do  not  swell  the  rods, 
II,  Subterminal  or  clostridial  spores, 

III,  Terminal  or  plec tridial  spores, 

a.  round  spores, 

b,  elongated  spores. 

Combining  Classes  I and  II  a total  of  fifty-three 
strains  are  included.  This  combined  class  is  separated  from 
Class  V on  the  basis  of  relation  to  combined  oxygen.  The  strains 
in  Classes  I and  II  showed  no  grov/th  in  the  closed  arm  of  the 
fermentation  tube  in  the  presence  of  dextrose.  Those  in  Class  V 


1 


01 

showed,  growth  under  suoh  conditions.  The  other  classes  (III,  IV, 
VI,  VII,  VIII,  IX,  X,  XI,  and  XII)  contains  only  one  strain  each, 
these  being  separated  from  the  other  larger  groups  on  the  basis 
of  characteristics  of  minor  importance,  such  as  action  on  starch, 
location  of  flagella,  etc. 


32 

IV.  Discussion  of  Results 

In  general  the  thermophilic  bacteria  constitute  a 
homogeneous  group  of  bacteria,  having  the  important  character- 

I 

I 

istics  of  bacterial  characterization  in  common. 

The  results  obtained  by  growing  some  of  these  thermo - 
philes  in  different  media  and  at  different  temperatures  agree 
with  those  obtained  by  Oprescu  (1896),  names  (1900),  Blau  (1906), 
and  Koch  and  Hoffman  (1911),  in  that  the  nature  of  the  medium 
seemed  to  influence  the  temperature  demanded  for  growth.  The 
examination  of  Table  111  snows  that  the  organisms  included  here 
grew  better  at  the  higher  temperatures  on  agar  slants  and  plates 
than  in  gelatin.  Some  of  those  which  showed  no  growth  at  all, 

or  only  slight  growth,  on  agar  slants  after  24  hours  incubation 

at  25°  - 30°  C.,  were  found  to  grow  well  in  gelatin  at  this 
temperature,  causing  some  liquefaction*  Of  course,  the  time 
must  be  taken  into  consideration  in  judging  the  growth  of  ther- 
mophiles  at  any  temperature.  It  was  found  that  some  strains 
which  showed  no  growth  on  agar  slants  at  room  temperature  in  24 

or  48  hours,  would  show  slight  growth  at  this  temperature  after 

a longer  inoubation.  Koch  and  Hoffman  (1911)  found  that  the 
organisms  they  studied  would  not  grow  in  artificial  media  at 
25°  - 28°  C.  but  did  not  mention  the  length  of  the  incubation 

period.  Probably  other  investigators  who  claimed  that  real 
thermophilic  organisms  would  not  grow  at  room  temperature  did 
not  take  into  consideration  the  time  required  for  growth  to  appear 


. 


. 


. 

. 


33 

at  this  temperature. 

Of  the  investigators  who  reported  thermal  death  points 
for  thermophilic  organisms,  Russell  and  Hastings  (1902)  and 
Bigelow  and  Hsty  (1920)  are  the  only  ones  who  described  their 
method  for  determining  them.  There  has  been  no  uniformity  in 
the  methods  used  for  determining  the  thermal  death  points  of 
thermophilic  bacteria.  Hence,  the  results  obtained  by  the 
different  investigators  cannot  very  well  be  compared  because  so 
many  factors  influence  thermal  death  points. 

In  comparing  the  results  obtained  in  this  investigation 
with  those  obtained  by  Bigelow  and  Bsty  (1920),  several  points 
deserve  mention.  The  strains  used  in  this  study  seemed  to  be 
less  resistant  to  moist  heat  than  those  used  by  the  above  men- 
tioned authors  under  the  conditions  which  obtained  in  this 
investigation.  Several  probable  explanations  can  be  offered  for 
this.  The  pH  of  the  broth  suspensions  of  spores  used  in  this 
investigation  ranged  from  7.4  to  8.0,  while  the  different  suspen- 
sions used  by  Bigelow  and  .feisty  ranged  from  6.0  to  6.3.  Ho 
attempt  was  made  to  adjust  the  pH  of  the  broth  after  the  spores 
had  been  grown  in  it,  and  no  change  was  noted  in  pH  during  the 
heating  of  the  suspensions,  except  in  a few  cases  where  the 
exposure  at  100°  G.  was  prolonged  beyond  70  minutes.  Probably 
if  the  pH  had  been  adjusted  to  7.0  of  even  6.0  it  would  have 
lengthened  the  time  required  to  destroy  the  spores  at  the  stated 
temperatures.  This  probability  has  suggested  to  the  author 
another  research  problem  in  connection  with  thermal  death  point 


, 


. 

. 

, • 


34 

determinations  of  thermophilic  bacteria.  She  hopes  to  be  able 
to  begin  work  on  this  problem  in  the  near  future. 

While  the  initial  concentration  of  spores  per  cc.  of 
suspension  determines  somewhat  the  time  required  for  their  com- 
plete destruction,  thermophiles  do  vary  in  the  degree  of  their 
resistance  to  heat,  This  is  demonstrated  by  the  data  given  in 
Table  IV,  The  suspension  for  culture  No,  10  contained  the  least 
number  of  spores  per  cc,  (390)  and  these  were  killed  by  a 15 
minute  exposure  at  100°  C,  Culture  89  (11,000  spores  per  cc.) 
required  am  exposure  of  180  minutes  at  100°  C,  for  complete 
destruction  while  culture  52  (73,000,000  spores  per  cc, ) only 
required  60  minutes  at  100°  0.  At  the  higher  temperatures  of 
115°  C.  and  120°  C*,  the  difference  in  time  required  to  kill  the 
spores  of  the  different  cultures  varied  only  0.5  to  1,0  minute 
and  0.25  to  1.0  minute,  respectively. 

The  spores  tested  by  Bigelow  and  Bsty  were  spores  of 
thermophilic  bacteria  isolated  from  cases  of  spoilage  in  a 
variety  of  canned  foods.  The  presence  of  these  organisms  in  the 
canned  foods  was  due  to  their  great  heat  resistance  and  ability 
to  withstand  the  usual  sterilizing  processes.  This,  no  doubt, 
explains  the  fact  that  these  spores  were  found  to  be  more  re- 
sistant to  heat  than  any  of  those  described  in  the  literature 
on  thermophiles.  The  spores  used  in  this  study  were  spores  of 
thermophilic  bacteria  isolated  from  wqter,  soil,  and  feces,  and 
had  been  grown  on  laboratory  media  for  12  months  or  more.  This 
calls  to  mind  a statement  made  by  Grieg-3mith  (1921)  to  the  effect 


35 

that  when  bacteria  have  been  recently  isolated  from  what  may  be 
called  their  natural  habitat,  they  may  be,  and  probably  are, 
more  vigorous  than  after  a spell  of  sub-cultivation  in  the  lab- 
oratory. 

Sames  (1900)  found  that  the  temperature  at  which  spores 
of  thermophilic  bacteria  were  produced  influenced  their  resist- 
ance to  flowing  steam  at  100°  C.  for  example,  the  spores  of 
one  organism  studied  by  him,  when  produced  at  56°  G.  resisted 
steam  at  100°  G.  for  120  minutes,  while  those  of  the  same  organ- 
ism, produced  at  37°  C.  withstood  the  same  conditions  only  for 
60  minutes.  The  spores  used  in  this  investigation  were  produced 
at  55°  C.  It  seems  quite  probable  that  the  temperature  at  which 
the  spores  are  produced  has  an  influence  on  their  resistant 
properties. 

Bahn  (1921)  says  it  is  well  known  that  temperature 
is  one  of  the  most  important  factors  of  life.  It  is  so  important 

that  the  higher  animals  protect  themselves  by  a very  complicated 
mechanism  of  regulation  against  changes  in  temperature;  the  life 
processes  of  such  animals  takes  place  at  a temperature  nearly 
constant  from  birth  till  death.  This  causes  the  growth  of  warm- 
blooded animals  to  be  different  from  all  other  organisms.  All 
other  organisms,  cold-blooded  animals  as  well  as  bacteria,  have 
the  temperature  of  their  environment,  and  the  decrease  in  tem- 
perature decreases  the  intensity  of  growth.  There  are,  of  course, 
limits  to  the  favorable  influence  of  high  temperatures.  Growth 


* 

, 


86 

of  bacteria  will  increase  with  rising  temperature  to  a certain 
point,  called  the  optimum  temperature.  The  highest  temperature 
at  which  growth  will  take  place  is  oalled  the  maximum  temperature. 
Correspondingly,  the  minimum  temperature  of  an  organism  is  the 
lowest  point  at  which  growth  can  take  place. 

The  relationships  of  bacteria  to  temperature  are 
varied.  Buchanan  (1921)  divided  bacteria  into  three  groups 
dependent  on  variations  in  their  optimum  temperatures: — 

» 

I.  Thermophilic  bacteria 

Optimum  usually  above  45®  - 50®  C. 

II.  Mesophilio  bacteria 

Optimum  20°  - 57.5°  0. 

III.  Psychrophilic  bacteria 

Optimum  usually  below  10®  C. 

He  says  of  thermophilic  bacteria,  that  the  minimum  of  most  true 
thermophiles  is  above  40®  C.,  and  the  maximum  temperature  of 
some  of  thermophilic  bacteria  is  nearly  80®  C. 

A review  of  the  thermophilic  bacteria  described  in  the 
literature  shows  that  the  r ange  at  which  these  organisms  have 
been  found  to  grow  is  very  great.  It  is  evident  then,  that  some 
division  must  be  made  in  this  group  of  bacteria.  Some  of  the 
terms  used  by  the  different  investigators  for  the  gradations  of 
organisms  generally  included  in  this  group,  are,  thermophiles, 
ortho thermophiles,  thermo -tolerants,  psychrotolerants,  obligate 
thermophiles,  true  thermophiles,  facultative  thermophiles,  and 
strict  thermophiles.  Some  divisions  have  been  made  on  the  basis 
of  the  minimum  temperature,  some  on  the  basis  of  the  maximum 


37 

temperature,  and  some  on  the  basis  of  the  optimum  temperature. 

The  question  then  arises  as  to  whether  it  is  necessary 
to  regard  these  bacteria  as  constituting  a separate  group,  if 
the  rapidity  of  their  growth  at  a certain  temperature  is  the  only 
characteristic  separating  them  from  other  bacteria  which  grow 
more  rapidly  at  the  same  temperature.  This  question  would  not 
arise  in  the  case  of  the  strict  or  true  thermophiles  --  those 
which  grow  best  at  55°  0.  and  above.  In  the  case  of  the  inter- 
grading forms,  however,  some  confusion  might  arise.  Similar 
situations  exist  in  other  phases  of  the  science,  and  it  is  the 
intergrading  form  which  has  caused  trouble  in  the  systematic 
arrangement  and  characterization  of  all  forms  of  life.  Patho- 
genicity is  a function  of  bacteria  which  has  received  much  study 
and  upon  which  bacteriologists  have  much  definite  information. 
Still,  there  are  many  semi -pathogenic  bacteria  acting  as  con- 
necting links  betY/een  the  pathogenic  members  and  the  non-patho- 
genic  members  of  a group,  for  instance,  in  the  colon-typhoid 
group  there  is  the  pathogenic  .bacterium  typhosum  on  one  end  and 
the  ordinarily  non -pathogenic  Bacterium  coli  on  the  other.  A 
similar  situation  may  exist  for  the  thermophilic  function,  if 
it  may  be  discussed  as  such  for  the  moment. 

It  would  seem  that  the  best  basis  for  a division  is 

the  optimum  temperature  for  growth,  since  it  has  been  proved 
that  by  acclimatization,  both  the  minimum  and  maximum  temperatures 
for  an  organism  can  be  either  raised  or  lowered.  The  author 


f 


« 


? 


28 

proposes  the  following  division  of  bacteria  into  groups,  using 
the  optimum  temperature  as  the  basis  for  the  division. 

I*  Strict  thermophiles 

Optimum  temperature  above  55°  C. 

II.  Facultative  thermophiles 

Optimum  temperature  50°  - 55°  0. 

III.  Thermo tolerant  baoteria 

Optimum  temperature  40°  - 50®  C. 

IV.  iw.esopnilic  bacteria 

Optimum  temperature  25°  - 40®  G. 

V.  Psychrotolerant  bacteria 

Optimum  temperature  lo®  - 25®  G. 

VI.  Facultative  psychrophiles 

Optimum  temperature  0®  - 10®  G.  (?) 

VII.  strict  psychrophiles 

uptimum  temperature  below  0®  C.  (?) 

It  is  evident  from  the  statements  in  the  text-books 
on  bacteriology  that  the  psychrophilic  bacteria  should  receive 
the  same  critical  analysis  that  the  thermophilic  bacteria  are 
receiving.  It  is  probable  that  many  of  the  statements  concern- 
ing psychrophilic  baoteria  have  been  handed  on  from  text-book  to 
text-book  and  that  they  do  not  rest  upon  adequate  experimental 
data.  It  is  interesting  to  note  that  the  halophilic  bacteria 
are  beginning  to  receive  analysis  and  that  it  is  probable  that 
our  information  on  these  bacteria  will  be  greatly  amplified  in 
the  near  future. 

hince  the  growth  of  thermophilic  bacteria  at  ordinary 
temperatures  seems  to  involve  a time  element,  it  is  probable 
that  canned  foods  that  contain  thermophilic  bacteria  might  spoil 


■ 


. 


after  being  stored  for  a long  time  at  ordinary  temperatures.  Of 
oourse,  if  stored  at  higher  temperatures  it  is  well  known  that 
spoilage  occurs  very  promptly. 

Several  other  problems  in  connection  with  thermophilic 
bacteria  have  been  suggested  to  the  author  by  the  present  invest- 
igation. These  will  be  taken  up  in  the  near  future. 


r 


* 


- 


- 


40 


V.  Summary 

1*  Eighty-nine  strains  of  thermophilic  bacteria, 
studied  according  to  the  index  number  as  expressed  on  the 
descriptive  chart  of  the  Society  of  American  Bacteriologists, 
fell  into  twelve  classes.  All  of  these  separations  are  based 
upon  unessential  characteristics  of  classification.  In  the 
light  of  this  fact,  the  thermophilic  group  seems  to  be  a homo- 
geneous one,  the  members  having  in  common  all  of  the  important 
characteristics  used  in  classification. 

2.  The  "Index  Number"  is  a distinct  improvement  over 
the  old  "Group  Number",  however,  some  of  the  points  in  the  "Index 
Number"  would  be  difficult  to  determine.  Eor  instance,  the  func- 
tion of  pathogenicity  could  not  be  determined  without  a great 
deal  of  experimental  work. 

3.  The  thermal  resistance  of  eighteen  strains  of 
thermophilic  bacteria  was  studied.  At  the  higher  temperatures, 
115°  0.  and  120°  G.,  the  thermal  death  points  of  these  organ- 
isms fell  within  narrower  time  limits  than  those  at  the  lower 
temperatures,  100°  C.,  105°  C.,  and  110°  C. 

4.  Thermal  death  points  of  bacteria  were  influenced 
by  the  number  of  spores  in  the  suspension. 

5*  The  character  of  the  media  influenced  the  temper- 
atures for  the  growth  of  thermophiles. 

6.  A tentative  grouping  of  bacteria,  according  to 
temperature  optimums,  has  been  proposed. 


- JL 


1 


r 


, - 


41] 

VI,  Bibliography 

Ambroz,  A*  1910  Uber  das  Phfinomen  der  Ihermobiose  bei  den 

Mikroorganismen.  Centralbl.  f.  Bakteriol.  Abt.I.  Ref., 
48,  257  and  289. 

Ambroz.  A«  1913  Denitrobakterium  therinophilum  spec,  nova,  ein 
Beitrag  zur  Biologie  der  thermopilen  Bakterien. 
Centralbl.  f.  Bakteriol.  Abt.II.,  37,  3. 

Anitschkow,  U.  U.  1906  Zur  Prage  fiber  die  Rolle  der  thermophiler 
Bakterien  im  Darmkanal  des  Mensohen.  Centralbl.  f. 
Bakteriol.  Abt.I.  Orig.,  41,  326  and  426. 

Arloing,  Cornevin,  et  Thomas  1882  Moyen  de  confe'rer  artifi- 

ciellement  1 * imnnmite^  contre  le  charbon  symptomatique 
on  bacte'rien  avec  du  virus  attenue7.  Compt.  rend.  Acad, 
d.  sc.,  95,  189. 

Baker,  H.  R.  1921  Substitution  of  brom-thymol-blue  for  litmus 

in  routine  laboratory  work.  Jour.  Bact.,  7,  301. 

✓ 

Bardou  1906  Etude  biochimique  de  quelques  Bacteriacees  ther- 
mophiles  et  de  leur  role  dans  la  disintegration  des 
matibres  organiques  des  eaux  d'egovt.  (Lille  1906.) 
Original  not  seen.  Reviewed  by  Ambroz,  A.  1910. 
Centralbl.  f.  Bakteriol.  Abt.I.  Ref.,  39,  744. 

Barlow  1912  A spoilage  of  canned  corn  due  to  a thermophilic 
bacterium.  Thesis  for  Degree  of  Master  of  Science, 
University  of  Illinois. 


. t 


Benignetti,  B.  1905  Di  un  germe  termofilo  isolato  dai  fanghi 

d*Acqui.  Riv.  d'lgiene  a Banita  pubbl.  1906.  Original 
not  seen.  Reviewed  in  Centralbl.  f.  Bakteriol.  Abt. II., 
14,  420. 

Bergey,  B • 1919  Thermophilic  bacteria.  Jour.  Baot.,  4,  301. 

Bigelow,  W.  B.  and  Esty,  J.  R.  1920  The  thermal  death  point  in 
relation  to  time  of  typical  thermophilic  organisms. 

Jour.  Infect.  Bis.,  27,  602. 

Blau,  0.  1906  Ueber  die  Temperaturmaxima  der  Sporenke  inning  und 

der  Bporenbildung,  sowie  die  supramaximalen  TOtungs- 
zeiten  der  Sporen  der  Bakterien,  auch  derjenigen  mit 
hohen  Tempera turminima.  Centralbl.  f.  Bakteriol.  Abt. 
II.,  15,  97. 

Brazzola,  P.  1906  Significata  dei  batteri  termofili,  di  quelli 
della  putrefazione  e del  gruppo  coli,  nell'esame 
batteriologico  delle  acque.  Centralbl.  f.  Bakteriol. 
Abt. II.,  16,  582. 

Bredfeld  1878  Untersuohungen  fiber  die  Spaltpilze,  Bacillus 
subtilis.  1878.  Original  not  seen.  Reviewed  by 
Mace”,  E.  1912  Traite  Pratique  de  Baeteriologie, 

v.  I.,  p.  97. 

Brewer,  W.  H.  1866  The  presence  of  living  species  in  hot  and 

saline  waters  in  California.  Amer.  Jour.  Be.  and  Arts, 
2nd  ser.,  41,  391. 

Bruini,  G.  1905  Ueber  die  thermophile  Mikrobenflora  des  men- 


I 


f 


t * 


f 


43 


schlichen  Darmkanals.  Centralbl.  f.  Bakteriol.  Abt. 

I.  Orig. , 38,  298. 

Buohanan,  E.  D.  and  Buchanan,  R.  E.  1921  Bacteriology  kao- 
Millan  Company,  Hew  lork. 

Buohanan,  R.  E.  1922  The  thermophilic  bacteria  in  canning. 

The  Canner,  54,  Bo.  22,  p.  1. 

Burrill,  T.  J.  1889  The  biology  of  ensilage.  Agr.  Exp.  Sta., 
Uni.  111.  Bull.  7,  177. 

Bushnell,  L.  D.  1922  Quantitative  determinations  of  some  of 
the  biochemical  changes  produced  by  a saphrophytic 
anaerobe.  Jour.  Bact.  7,  373. 

Gambler,  R.  1896  Resistance  des  germes  baoteriens  a la  chaleur 
seohe.  Ann.  de  Micr.,  8,  49. 

Gatterina,  G.  1904  Beitrag  zum  Studium  der  thermophilen 

Bakterien.  Centralbl.  f.  Bakteriol.  Abt. II.,  12,  353. 

Certes,  A.  et  Garrigou  1886  Be  la  presence  constants  de  micro - 
organismes  dans  les  eaux  de  luohon,  recueillies  au 
griffon  b la  temperature  de  64°,  et  de  leur  action 
sur  la  production  de  la  barekine.  Compt.  rend.  Acad, 
d.  sc.  103,  70S. 

Gheyney,  E.  W#  1919  Study  of  micro-organisms  found  in  mer- 
chantable canned  foods.  Jour.  Med.  Research,  40,  177. 

Cohn,  E.  1876  Untersuohungen  tiber  bacterien.  Beit.  Biol. 

Pflan. , 2,  271. 

Cohn,  E.  1890  Ueber  die  WSrmeerzeugung  durch  Schimmelpilze  u. 

Bakterien  Yortrag,  gehalten  etc.  zu  Brieg  a.  15. /6. 


• 4 


44 

1890;  ref.  in  Kochs  Jahresb.,  1890,  Bd.  1,  S.  45. 
Original  not  seen.  Reviewed  by  Lafar-Handb.  d.  Tech. 
Mykol.,  3,  421. 

Cohn,  *'•  1893  Ueber  thermogene  Baoterien.  Ber.  d.  deutsch. 

Bot.  Gesellsoh.,  11,  (66). 

Conn,  H.  J.  et  al  1920  Report  of  the  committee  on  the 

descriptive  chart  for  1919.  Jour.  Bact.  5,  127. 
Davenport,  C.  B.  and  Castle,  W.  E.  1895  Acclimatization  of 

organisms  to  high  temperatures.  Arch.  f.  Entwcklngs- 
mechn.  d.  Organ.,  2,  227. 

Davis,  B.  M.  1897  Vegetation  of  the  hot  springs  of  Yellowstone. 
So.,  6,  145. 

de  Kruyff,  E.  1910  Les  Bacteries  thermophiles  dans  les  Tropiques 
Centralbl.  f.  Bakteriol.  Abt.II.,  26,  65. 

Donk,  P.  J.  1920  A highly  resistant  thermophilic  organism. 

Jour.  Bact.  5,  373. 

Dupont,  C.  1902a  Sur  les  fermentations  aerobies  du  fumier. 

Compt.  rend.  Acad.  d.  sc.  134,  1449. 

Dupont,  C.  1902b  Sur  les  fermentations  aBrobies  du  fumier  de 
ferrne.  Ann.  Agron.,  28,  289. 

Ehrenberg  1858  Monatsberichte  d.  k.  P.  Akad.  Wiss.  Berl.  1858. 

S.  488.  Original  not  seen.  Reviewed  by  Ambroz  1910. 
Paloioni,  D.  1907  I germi  termofili  nelle  aoque  del  Bullieame. 

Arch,  di  farmacol.  sperim. , 1907,  Do.  1.  Original  not 
seen.  Reviewed  in  Centralbl.  f.  Bakteriol.  Abt.  II., 

20,  164. 


f f • 


* » 


f * 


i * 


4 I 


45 

Plourens  1846  Compt.  rend.  Acad.  d.  sc.  23,  934. 

Pltlgge,  C.  1894  Die  Aufgaben  und  Leistungen  der  kilchsterili- 
sirrung  gegentlber  den  Darmkrankheiten  der  Slfuglinge. 
Ztschr.  f.  Hyg.  u.  Infectionskrankh. , 17,  273. 

Georgevitch,  P.  1910a  Bacillus  thermophilus  vranjensis.  Arch, 
f.  hyg. , 72,  201. 

Georgevitch,  P.  1910b  Bacillus  thermophilus  Jivofni  nov.  spec. 

und  Bacillus  thermophilus  Losanitchi  nov.  spec.  Central- 
bl.  f.  Bakteriol.  Abt.II,  27,  150. 

Gilbert,  Dr.  1904  Ueber  Actinomyces  thermophilus  und  andere 
Aotinomyoeten.  Ztschr.  f.  Hyg*  u.  Infectionskrankh. , 

47,  383. 

Globig,  Dr.  1888  Ueber  Bakterien-Wachstum  bei  50°  bis  70°  0* 
Ztschr.  f.  Hyg.  u.  Infectionskrankh.,  3,  394. 

Gorini  1895  Studi  critico-sperimentali  sulla  sterilizzazione 

del  latte.  Giornale  della  reale  Societk  d*Igiene.  1895 
Ho.  1.  Original  not  seen.  .Reviewed  by  Ambroz,  1910. 

Grieg-Bmith,  R.  1921  The  high  temperature  organism  of  fer- 
menting tan-bark.  Part  I.  Proc.  Linnean  Soc.  Hew 
South  Wales,  46,  Part  I.,  76. 

Griffiths,  A.  B.  1894  Microbes  involved  in  the  ensilage  of 
green  fodder;  and  on  the  variations  of  sugar  and 

acidity  with  temperature  and  time.  Chemical  Hews, 

70,  273. 


• t 


* 


■ ; c • 


46 

Hall,  I.  C.  1922  Differentiation  and  identification  of  the 
sporulating  anaerobes*  Jour,  infeot.  Die.  30,  445. 
Harding,  H.  a.  1896  Thesis  for  Degree  of  Bachelor  of  Arts, 

University  of  Wisconsin.  Original  not  seen.  Personal 
interview  with  author. 

Harshbarger,  J.  W.  1897  The  vegetation  of  Yellowstone  hot 
springs.  Am.  Jour.  Pharm. , 69,  625. 

Jttger  1909  Die  Bakteriologie  des  tttglichen  Lebens.  (Hamburg 
und  Leipzig,  1909)  Original  not  seen.  Reviewed  by 
Ambroz,  1910. 

Karlinski,  J.  1895  Zur  Kenntnis  der  Bakterien  der  Thermalquellen 

c 

Hyg.  Rundshau.  6,  685. 

A 

Kedzoir,  D.  1896  ueber  eine  thermophile  Cladrothrix.  arch.  f. 
Hyg.,  27,  328. 

Kehler,  W.  1904  Ueber  Methoden  zur  sterilisation  von  urdboden 
und  Pflanzensamen  und  liber  zwei  neue  thermoresistente 
Bakterien.  Dissertation-kBnigsberg,  1904. 

Koch,  R.  1876  Untersuchungen  iiber  Baxterien.  Beit.  Biol.  Pflan. 
2,  277. 

Koch,  A.  and  Hoffmann,  0.  1911  Uber  die  Verschiedenheit  der 

Tempera turspruche  thermophiler  Bakterien  in  Boden  und 
in  kunstlichen  Uahrsubstraten.  Gentralbl.  f.  Bakteriol. 
Abt.II . , 31,  433. 

Koning,  G.  J.  1897a  De  gisting  onzer  inlandsche  tabak.  Tijdschr. 

voor  toegepaste  scheikunde  en  hygiene.  Deel.  I.  1897. 
Original  not  seen.  Reviewed  by  Lafar,  P.  nandb.  d. 


47 

techn.  Mykologie,  6,  1.  from  Behrens,  J.  1906 
Mykologie  der  Tabakfabrikation. 

Zoning,  0,  J,  1897b  HollancLsche  tabak.  Be  Ratuur,  189  7. 

Original  not  seen.  Reviewed  by  Lafar,  i*'.  handb.  d. 
techn.  Mykologie,  5,  1.,  from  Behrens,  J.  1905  Mykologie 
der  Tabakfabrikation. 

Zoning,  C.  J.  1898  Hollandsche  tabak  II.  Be  Uatuur  1898 

Original  not  seen.  Reviewed  by  Lafar,  B.  Handb.  d. 
techn.  mykologie,  5,  1.,  from  Behrens,  J.  1905 
Mykologie  der  Tabakfabrikation. 

Kossowicz,  A.  1912  Mykologisehe  und  warenkundliche  Lotizen. 

(2.  Hitteilung. ) Ztschr.  f.  Landw.  Versuchsw. , 15,  757. 
Kossowicz,  A.  1915  Occurrence  of  thermophilic  Bacteria. 

Deutsch.  Zuckerind.,  37,  1019.  Original  not  seen. 
Reviewed  in  Chem.  Absts.,  7,  2699. 

M 

Kroulik,  A.  1912  Bber  thermophile  zellulosevergfirer.  Centralbl. 

f.  Bakteriol.  Abt.II.,  36,  339. 
laxa,  0.  1898  Ueber  einen  thermophilen  Bacillus  aus  Zucker- 

fabriksprodukten.  Centralbl.  f.  Bakteriol.  Abt.II., 

4,  362. 

«• 

leichman  1893  Uber  eine  schleimige  Gfirung  der  Milch,  land- 
wirtsch.  Yers. -Station. , Berl.,  43,  375. 

McFadyen,  A.  and  Blaxall,  IP.  R.  1894  Thermophilic  bacteria. 

Jour.  Bath,  and  Bacteriol.,  3,87. 

McBadyen,  A.  and  Blaxall,  jp.  R.  1896  Thermophilic  bacteria. 

Brit.  Med.  Jour.,  2,  644. 


» a 


* » 


48 


Miehe,  H.  1907a  Die  Selbsterhitzung  des  Heuee.  Original  not 
Been.  Reviewed  in  Centralbl.  f.  Bakteriol.  Abt.II., 
20,  295. 

Miehe,  H.  1907b  Thermo I dium  sulfureum  n.  g.  n.  sp.,  ein  neuer 
Wfirmepilz.  Ber.  d.  deutech.  Bot.  Gesellsch.,  25,  510. 

Miquel,  P.  1897  Bull,  de  la  Statisque  municipale  de  Paris, 

Dec.  1897.  Original  not  seen.  Reviewed  by  Tsiklinsky, 
Mile.  P.,  1899b.  Ann.  de  l'Inst.  Pasteur.  15,  788. 

Miquel,  P.  1881  Annuaires  de  Montsouris,  1881,  464.  Original 
not  seen.  Reviewed  by  Mace',  E.  1912  Traite  Pratique 
de  Bacteriologie,  1,  97. 

Miquel,  P.  1682  Les  organismes  vivants  de  1 'atmosphere . Ih&se 
de  Paris.  Original  not  seen.  Reviewed  by  Mace,  E. 

1912  Traite  Pratique  de  Bacte'riologie,  1. 

Miquel,  P.  1888  Monographie  d'un  Bacille  vivant  au-dela  de 
70°  0.  Ann.  de  Mior.,  1,  4. 

Michaelis,  G.  1899  Beitrage  zur  Kenntnis  der  thermophilen 
Bakterien.  Arch.  f.  Hyg.  26,  285. 

Miyoshi,  M.  1897a  Ueber  das  massenhafte  Vorkommen  von  Eisen- 
bakterien  in  den  Thermen  von  Ikao.  Jour.  Coll.  Sc. 

Imp.  Univ.  Tokyo,  10,  129.  Original  not  seen.  Review- 
ed in  Centralbl.  f.  Bakteriol.  Abt.II.,  2,  526. 

Miyoshi,  M.  1897b  Studien  fiber  die  Schwefelrasenbildung  und 

die  Schwefelbakterien  der  Ihermen  von  Xumoto  bei  Dikko. 
Jour.  Coll.  Sc.  Imp.  Univ.  Tokyo,  10,  142.  Original 


49 


not  seen*  Reviewed  in  Gentralbl*  f*  Bakteriol,  Abt.II, 
3,  325. 

Negre,  L.  1913a  Baoteries  thermophiles  des  sables  du  Sahara* 
Compt.  rend*  Soo*  de  biol.,  74,  814* 

Negre.  L*  1913b  Bacteries  thermophiles  des  eaux  de  Piguig. 

Compt.  rend.  Soc*  de  biol.,  74,  867. 

Noack,  K.  1912  Beitrflge  zur  Biologie  der  thermophilen  Organ- 
ismen.  Dissertation,  Leipzig,  1912. 

Norton,  J.  P.  and  Sawyer,  Mary  V.  1921  Indol  production  by 

bacteria.  Jour.  Bact.  6,  471. 

Oprescu,  Y.  1898  Studien  fiber  thermophile  Bakterien.  Arch.  f. 
Hyg.,  33,  164. 

Patzschke,  W.  1919  ttber  die  Widerstandsfflhigkeit  von  Bakterien 
gegentiber  hohen  Temper  a turen  und  das  Lobecksche 
Biorisierverfahren.  Ztschr.  f.  Hyg.  81,  226. 

Plimmer,  H.  0.  and  Paine,  S.  G-.  1921  A new  method  for  the 

staining  of  bacterial  flagella.  Jour.  Path,  and 
Bacteriol.  24,  286. 

Poupe,  Pr.  1897  BBhem.  Zeitschr.  1897/8,  Bd.  22,  S.  341. 

Original  not  seen.  Reviewed  by  Lafar,  P.,  Handb.  d. 
teohn.  Mykologie  v.XI,  p.  469. 

Pringsheim,  H.  1911  fiber  die  Assimilation  des  Luftstickstoffs 
durch  thermophile  Bakterien.  Gentralbl.  f.  Bakteriol. 
Abt.II. , 31,  23. 

fl 

Pringsheim,  H.  1913  Bber  die  Yergfirung  der  Zellulose  durch 


50 


thermophile  Bakterien.  Centralbl.  f.  Bakteriol.  Abt. 
II. t 38,  513. 

Rabinowitsch,  L.  1895  Ueber  die  thermophilen  Bakterien.  Ztschr. 
f.  Hyg.#  20,  154. 

Rahn,  0.  1921  In  Marshall’s  Microbiology,  P.  Blakiston’s  Son 

& Co. 

Russell,  H.  L.  and  Hastings,  E.  G.,  1902  A Micrococcus,  the 

thermal  death  limit  of  which  is  76°  C.  Centralbl.  f. 
Bakteriol.  Abt.II.,  8,  339. 

Sames,  T.  1900  Zur  Kenntnis  der  bei  hOherer  Tempera turen 

wachsenden  Bakterien  und  Streptothrixarten.  Ztschr. 
f.  Hyg.  33,  313. 

n 

Sohardinger,  3?.  1903  Uber  thermo-chile  Bakterien  aus  verschied- 

enen  Speisen  und  Milch.  Ztschr.  f.  Untersuch.  d. 
Hahrungs-u.  Genus s -mitt el,  6,  865. 

Sohillinger,  A.  1898  Ueber  thermophilen  Bakterien.  Hyg. 
Rundschau,  8,  568. 

Schloesing,  Th.  fils  1888  Sur  la  combustion  lente  de  certaines 
matieres  organiques.  Compt.  rend.  Acad.  d.  sc.  106, 
1293. 

Schloesing,  Th.  fils  1889  Sur  la  combustion  lente  de  certaines 
matieres  organiques.  Compt.  rend.  Acad.  d.  so.  108, 

527. 

Schloesing,  Th.  et  fils.  1892  Contribution  a 1* etude  des  fer- 
mentations du  fumier.  Ann.  Agron.,  16,  5. 

Schtltze,  H*  1908  Beitr&ge  zur  Kenntnis  der  thermophilen 


• ♦ 


51 


Aotinomyzeten  und  ihrer  Sporenbildung.  Arch.  f.  Hyg. 
67,  35. 

Sohwabe  1837  liber  die  Algen  der  Karlsbader  warmen  Quellen. 
Linnaea  11,  109. 

Setchell,  W.  A.  1903  The  upper  temperature  limits  of  life. 

So.,  17,  934. 

Sonnerat  1774  Observation  d'un  phenomena  singulier  sur  des 
poissons  qui  vivent  dans  une  eau  qui  a 69°  chaleur. 
Jour,  de  Physique.  T.  3.1774.  p.  256.  Original  not 
seen.  Reviewed  by  Ambroz)1910. 

Tanner,  ?.  W.  and  Back,  G.  M.  1922  Clostridium  botulinum. 

Jour.  Infect.  Die.  31,  92. 

Teioh,  M.  1896  Beitrag  zur  Kenntnis  thermophiler  Bakterien. 

Hyg.  Rundschau,  6,  1094. 

Tirelli,  E.  1907  I termofili  delle  aoque  potabili.  Riforma  med. 
10,265.  Original  not  seen.  Reviewed  in  Gentralbl. 
f.  Bakteriol.  Abt.II.,  19,  328. 

Tsiklinsky,  Mile.  P.  1898  0 mikrobach  jiwuschieh  pri  visokieh 

temperaturaoh.  Russ.  Arch.  f.  Pathol,  etc.  Bd.  V.  1898 
Original  not  seen.  Reviewed  by  Ambroz  1910. 

Tsiklinsky,  Mile.  P.  1899a  Sur  les  mucedinees  thermophiles. 

Ann.  de  l'Inst.  Pasteur  13,  500. 

Tsiklinsky,  Mile.  P.  1899b  Sur  les  microbes  thermophiles  des 
sources  thermales.  Ann.  de  l'Inst.  Pasteur  13,  788. 
Tsiklinsky,  Mile.  P.  1903  Sur  la  flore  microbienne  thermophile 

du  canal  intestinal  de  l'homme.  Ann,  de  l'Inst.  Pasteur 


52 


17,  217 • 

Van  Tieghem,  M.  P.  1881  Sur  des  bacteriacees  vivant  a la 

temperature  de  74°  centigr.  Bull,  Boo,  bot.  de  Prance 
28,  35. 

Yelich,  A.  1914  t)ber  thermophile  Mikroorganismen.  Gaspois 

ceskych  lfckarur.  53,  1914,  p.  1026.  Original  not  seen. 
Reviewed  in  Centralbl.  f.  Bakteriol.  Abt.II.,  44,  174. 
Vernhout,  J.  H.  1898  Rapport  over  het  bacteriologisch  onderzoek 
van  gefermenteerde  tabak.  Tesjsmannia.  Jahrg.  1898, 

Nr.  2/3.  Original  not  seen.  Reviewed  by  Lafar,  P. 
Handb.  d.  techn.  Mykologie,  5,  1.  from  Behrens,  J. 

1905  Mykologie  der  Tabakfabrikation. 

Vemhout,  J.  H.  1899  Onderzoek  van  baoterien  bij  de  fermentatie 
der  tabak.  Mededeelingen  iut  s 'Lands  Plantentium. 

1899.  XXXIV,  Original  not  seen.  Reviewed  by  Lafar,  P, 

v 

Handb.  d.  teohn.  Mykologie,  5,  1,  from  Behrens,  J. 

1905  Mykologie  der  Tabakfabrikation. 

Weber  1895  Die  Baoterien  der  sogenannten  sterilen  Milch  des 
Handels  usw.  Arb.  a.  d.  Kaiserl.  Gesundheitsamte. 

Bd.  XVII.  1895.  p.  108.  Original  not  seen.  Reviewed 
by  Ambroz  1910. 

Weinzirl,  J.  1919  The  bacteriology  of  canned  foods.  Jour. 

Med.  Research  39,  349. 

Winslow,  G.-E.  A.,  et  al  1920  The  families  and  genera  of  the 

bacteria.  Pinal  report  of  the  Gommittee  of  the  Society 
of  American  Bacteriologists  on  Gharacterization  and 


• r 


53 


Wittlin, 


Classification  of  bacterial  types.  Jour.  Bacteriol., 
5,  191. 

J.  1897  Bakteriologische  Untersuchung  der  Mineral- 
quellen  der  Schweiz  II  Thermen  die  fhermalquellen  in 
Ragaz-Pf&fers  (Kant on  St.  Oallen)  Original  not  seen. 
Reviewed  in  Centralbl.  f.  Bakteriol.  Abt.Ii,  3,  4u0. 


. 


* ' 

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Biographical  Sketch 


Lethe  Uleanora  Morrison  received  her  common  school 
education  in  the  common  schools  of  Waterloo,  Illinois,  She 
was  graduated  from  Waterloo  High  Sohool  in  1910,  She  taught 
in  the  common  schools  of  Illinois  from  1911  to  1914,  In  the 
fall  of  1914  she  entered  the  University  of  Illinois,  Urbana, 
Illinois,  and  in  June,  1919,  received  the  degree  of  Bachelor 

of  Arts,  having  taken  a scientific  course  with  home  economics 
as  the  ma^or  study. 

She  entered  upon  duties  as  half-time  assistant  in 
bacteriology  in  the  Bacteriology  Department,  University  of 
Illinois  in  1919,  In  the  fall  of  the  next  year  she  accepted 
a full  time  assistantship  in  the  same  department.  During  the 
tenure  of  these  two  assistantships,  graduate  work  was  taken  in 
the  Graduate  Sohool  of  the  University,  The  degree  of  Master  of 
Science  in  Bacteriology  was  received  from  this  institution  in 
June,  1921,  Prom  1921  to  1922  she  was  a fellow  in  Bacteriology 
in  the  University  of  Illinois, 

She  is  a member  of  the  Society  of  American  Bacteri- 
ologists and  the  American  Public  Health  Association.  She  is  also 
a member  of  Omicron  Hu,  Iota  Sigma  Pi,  and  an  active  member  of 
Sigma  Xi. 

Publications 

Studies  on  Thermophilic  Bacteria  I,  Aerobic 
Thermophilic  Bacteria  from  Water,  with  Pred  W.  Tanner.  Journal 


,L  : 


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